Human Dickkopf-related protein and nucleic acid molecules and uses therefor

ABSTRACT

Novel Dkk and Dkk-related polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length Dkk and Dkk-related proteins, the invention further provides isolated fusion proteins, antigenic peptides and antibodies. The invention also provides Dkk and Dkk-related nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a Dkk and Dkk-related gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/069,137, filed Feb. 28, 2005, now U.S. Pat. No. 7,645,451, which is acontinuation of U.S. patent application Ser. No. 09/972,473, filed Oct.4, 2001, now U.S. Pat. No. 7,057,017, which is a continuation of U.S.patent application Ser. No. 09/263,022, filed Mar. 5, 1999 (abandoned),which is a continuation-in-part of PCT Application No. PCT/US98/07894designating the U.S.A., filed Apr. 16, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 09/009,802,filed Jan. 20, 1998 (abandoned), which claims the benefit of U.S. PatentApplication No. 60/071,589, filed Jan. 15, 1998 (abandoned), and is acontinuation-in-part of U.S. patent application Ser. No. 08/842,898,filed Apr. 17, 1997 (abandoned), which is a continuation-in-part of U.S.patent application Ser. No. 08/843,704, filed Apr. 16, 1997 (abandoned).The contents of each of the above-referenced patent applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Secreted proteins play an integral role in the formation,differentiation, and maintenance of cells in multicellular organisms.For instance, secretory proteins are known in the art to be involved insignaling between cells which are not in direct contact. Such secretedsignaling molecules are particularly important in the development ofvertebrate tissue during embryogenesis as well as in the maintenance ofthe differentiated state of adult tissues. For example, inductiveinteractions that occur between neighboring cell layers and tissues inthe developing embryo are largely dependent on the existence andregulation of secreted signaling molecules. In inductive interactions,biochemical signals secreted by one cell population influence thedevelopmental fate of a second cell population, typically by alteringthe fate of the second cell population. For example, the Wnt proteinsare now recognized as one of the major families of developmentallyimportant signaling molecules in organisms ranging from Drosophila tomice.

The Wnt gene family encode a large class of secreted proteins related tothe Int1/Wnt1 proto-oncogene and Drosophila wingless (“Wg”), aDrosophila Wnt1 homologue, (Cadigan et al. (1997) Genes & Development11:3286-3305). Wnts are expressed in a variety of tissues and organs andare required for many developmental processes, including segmentation inDrosophila, endoderm development in Caenorhabditis elegans,establishment of limb polarity, neural crest differentiation, kidneymorphogenesis, sex determination, and brain development in mammals(reviewed in Parr and McMahon (1994) Curr. Opinion Genetics & Devel.4:523-528; Cadigan and Nusse, supra).

Recent studies in diverse organisms have led to identification ofseveral components of the Wnt signal transduction pathway in respondingcells (Cadigan and Nusse, supra). Wnt signals are transduced by theFrizzled (“Fz”) family of seven transmembrane domain receptors (Bhanotet al. (1996) Nature 382:225-230). The resulting signal leads to theactivation of the cytoplasmic protein Dishevelled (Dsh) andstabilization of Armadillo/β-catenin (Perrimon (1994) Cell 76:781-784).Negative regulators of the Wnt pathway include glycogen synthase kinase3 (GSK3)/shaggy (Perrimon, supra), the tumor suppressor gene productadenomatous polyposis coli (APC) (Gumbiner (1997) Curr. Biol.7:R443-436) and a novel protein, called Axin (Zeng et al. (1997) Cell90:181-192). In the absence of a Wnt ligand, these proteins promotephosphorylation and then degradation of β-catenin, whereas Wnt signalinginactivates GSK3, thus preventing β-catenin degradation. As a result,β-catenin is translocated to the nucleus, where it forms a complex withTCF transcription factors and activates target gene expression (Cadiganand Nusse, supra). Deregulation of this pathway can lead tocarcinogenesis (reviewed by Gumbiner, supra), emphasizing thelong-recognized connection between Wnts, normal development and cancer.This connection has been further established recently with theidentification the c-Myc protooncogene as a target of Wnt signaling (Heet al. (1998) Science 281:1509-3512).

While the outcome of Wnt signaling may be influenced by multipleintracellular regulatory mechanisms, recent studies have identifiedseveral classes of secreted factors which can modulate Wnt actionoutside of the cell. These include Cerberus, a secreted Wnt inhibitorimplicated in head development (Bouwmeester et al. (1996) Nature382:595-601), and a family of proteins related to the extracellulardomain of Frizzled. These Frizzled-related proteins (“FRPs”) (Rattner etal. (1997) Proc. Natl. Acad. Sci. USA 94:2859-2863), also known assecreted apoptosis-related proteins (“SARPs”), are encoded by severalindependently discovered genes including FrzA/FRP1, SDF5/FRP2,FrzB/FRP3, FRP4 and Sizzled (Melkonyan et al. (1997) Proc. Natl. Acad.Sci. USA 94:13636-13641; Finch et al. (1997) Proc. Natl. Acad. Sci. USA94:6770-6775; Wang et al. (1997) Cell 88:747-766; Leyns et al. (1997)Cell 88:747-756; Mayr et al. (1997) Mech. Dev. 63:109-325; and Salic etal. (1997) Development 124:4739-4748). These proteins inhibit theability of Xwnt8 to induce a secondary axis in frog embryos (for reviewsee Zorn (1997) Curr. Biol. 7:R501-504), and are thought to compete forbinding of Wnt ligands to the Frizzled receptors. Data on binding ofcertain FRPs to Xwnt8 (Wang et al., (1997) Biochem. Biophys. Res. Comm.236:502-504; and Leyns et al., supra) and Wg corroborate this notion(Rattner et al., supra).

It is now recognized that many of these families of signaling moleculeshave a dual role to play in both the development of an organism as wellas in promoting or maintaining the differentiated state of tissues inthe adult animal. Furthermore. major families of signaling moleculeshave been implicated in controlling proliferation of cells in matureadult tissue, for example, during normal cell turnover in the adultorganism as well as in tissue regeneration activated as a result ofdamage to the adult tissue. Given the important role of these signallingmolecules such as the Wnts and FRPs in both developing and adulttissues, there exists a need for identifying novel modulators of suchmolecules for use in regulating a variety of cellular processes.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnucleic acid molecules which encode a novel family of secreted humanproteins, referred to herein as the human Dickkopf proteins or “hDkks”(formerly referred to as the “Cysteine-Rich Secreted Proteins”, “CRSPs”,“CRISPYs”, or “CRSP proteins). The Dkk molecules of the presentinvention are useful as modulating agents in regulating a variety ofcellular processes. Accordingly, in one aspect, this invention providesisolated nucleic acid molecules encoding Dkk proteins or biologicallyactive portions thereof, as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of Dkk-encodingnucleic acids. In another aspect, this invention provides isolatednucleic acid molecules encoding Dkk-related proteins (e.g., Soggyproteins) or biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of Dkk- or Soggy-encoding nucleic acids.

In one embodiment, a Dkk nucleic acid molecule is 60% homologous to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98452 or complement thereof. In yet another embodiment,a Dkk nucleic acid molecule is 80% homologous to the nucleotide sequenceshown in SEQ ID NO:4, SEQ ID NO:6, or a complement thereof. In yetanother embodiment, a Dkk nucleic acid molecule is 60% homologous to thenucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:9, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98633, or a complement thereof. In yet anotherembodiment, a Dkk nucleic acid molecule is 85% homologous to thenucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:9, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98633, or a complement thereof. In yet anotherembodiment, a Dkk nucleic acid molecule is 70% homologous to thenucleotide sequence shown in SEQ ID NO:20. SEQ ID NO:22, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140, or a complement thereof. In yet anotherembodiment, a nucleic acid molecule of the present invention (e.g., aDkk-related nucleic acid molecule) is 90% homologous to the nucleotidesequence shown in SEQ ID NO: 13, SEQ ID NO: 15, or a complement thereof.

In a preferred embodiment, an isolated Dkk nucleic acid molecule has thenucleotide sequence shown SEQ ID NO:3, or a complement thereof. Inanother embodiment, a Dkk nucleic acid molecule further comprisesnucleotides 1-37 of SEQ ID NO: 1. In yet another preferred embodiment, aDkk nucleic acid molecule further comprises nucleotides 1088-2479 of SEQID NO:1. In another preferred embodiment, an isolated Dkk nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:1.

In another preferred embodiment, an isolated Dkk nucleic acid moleculehas the nucleotide sequence shown SEQ ID NO:6, or a complement thereof.In another embodiment, a Dkk nucleic acid molecule further comprisesnucleotides 1-124 of SEQ ID NO:4. In yet another preferred embodiment, aDkk nucleic acid molecule further comprises nucleotides 797-848 of SEQID NO:4. In another preferred embodiment, an isolated Dkk nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:4.

In another preferred embodiment, an isolated Dkk nucleic acid moleculehas the nucleotide sequence shown SEQ ID NO:9, or a complement thereof.In another embodiment, a Dkk nucleic acid molecule further comprisesnucleotides 1-108 of SEQ ID NO:7. In yet another preferred embodiment, aDkk nucleic acid molecule further comprises nucleotides 907-1536 of SEQID NO:7. In another preferred embodiment, an isolated Dkk nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:7.

In another preferred embodiment, an isolated Dkk nucleic acid moleculehas the nucleotide sequence shown SEQ ID NO:22, or a complement thereof.In another embodiment, a Dkk nucleic acid molecule further comprisesnucleotides 1-723 of SEQ ID NO:20. In yet another preferred embodiment.a Dkk nucleic acid molecule further comprises nucleotides 1501-3687 ofSEQ ID NO:20. In yet another preferred embodiment, an isolated Dkknucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:20.

In another preferred embodiment, an isolated nucleic acid molecule ofthe present invention (e.g., a Dkk-related nucleic acid molecule) hasthe nucleotide sequence shown SEQ ID NO:15, or a complement thereof. Inanother embodiment, a nucleic acid molecule further comprisesnucleotides 1-74 of SEQ ID NO: 13. In yet another preferred embodiment,a nucleic acid molecule further comprises nucleotides 801-928 of SEQ IDNO: 13. In yet another preferred embodiment, an isolated nucleic acidmolecule has the nucleotide sequence shown in SEQ ID NO:13.

In another embodiment, a Dkk nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence sufficientlyhomologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQID NO:8. or SEQ ID NO:21. In another preferred embodiment, a Dkk nucleicacid molecule includes a nucleotide sequence encoding a protein havingan amino acid sequence at least 60% homologous to the amino acidsequence of SEQ ID NO:2. In yet another preferred embodiment, a Dkknucleic acid molecule includes a nucleotide sequence encoding a proteinhaving an amino acid sequence at least 60% homologous to the amino acidsequence of SEQ ID NO:5. In yet another preferred embodiment, a Dkknucleic acid molecule includes a nucleotide sequence encoding a proteinhaving an amino acid sequence at least 60% homologous to the amino acidsequence of SEQ ID NO:8. In yet another preferred embodiment, a Dkknucleic acid molecule includes a nucleotide sequence encoding a proteinhaving an amino acid sequence at least 75% homologous to the amino acidsequence of SEQ ID NO:8. In yet another preferred embodiment, a Dkknucleic acid molecule includes a nucleotide sequence encoding a proteinhaving an amino acid sequence at least 65% homologous to the amino acidsequence of SEQ ID NO:21. In another embodiment, a nucleic acid moleculeof the present invention (e.g., a Dkk-related nucleic acid molecule)includes a nucleotide sequence encoding a protein having an amino acidsequence sufficiently homologous to the amino acid sequence of SEQ IDNO:14 (e.g., encodes a protein having an amino acid sequence which is60% homologous to the amino acid sequence of SEQ ID NO:14).

In another embodiment, an isolated nucleic acid molecule of the presentinvention encodes a Dkk protein which includes a signal sequence and atleast one cysteine-rich region, and is secreted. In another embodiment,an isolated nucleic acid molecule of the present invention encodes a Dkkprotein which includes a signal sequence and a cysteine-rich region,wherein the cysteine-rich region comprises at least one cysteine-richdomain, and is secreted. In yet another embodiment, a Dkk nucleic acidmolecule encodes a Dkk protein and is a naturally occurring nucleotidesequence.

In another embodiment, an isolated nucleic acid molecule of the presentinvention encodes a Dkk-related protein (e.g., a Soggy protein) whichincludes a signal sequence, lacks cysteine-rich domains, and issecreted. In another embodiment, an isolated nucleic acid molecule ofthe present invention encodes a Dkk-related protein (e.g., a Soggyprotein) which includes a signal sequence and a Soggy domain, and issecreted. In yet another embodiment, a nucleic acid molecule of thepresent invention encodes a Dkk-related protein and is a naturallyoccurring nucleotide sequence.

Another embodiment of the invention features nucleic acid moleculeswhich specifically detect Dkk nucleic acid molecules relative to nucleicacid molecules encoding non-Dkk proteins (or specifically detectDkk-related nucleic acid molecules). For example, in one embodiment, anucleic acid molecule hybridizes under stringent conditions to a nucleicacid molecule consisting of nucleotides 470-2479 of nucleotide sequenceshown in SEQ ID NO: 1, to nucleotides 1-475 of nucleotide sequence shownin SEQ ID NO:4. or to nucleotides 1-600 of nucleotide sequence shown inSEQ ID NO:7, or hybridizes under stringent conditions to the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98452, to the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or to thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140. In another embodiment, the nucleic acidmolecule is at least 500 nucleotides in length and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQID NO: 13, or SEQ ID NO:20 or a complement thereof.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a Dkk nucleic acidor Dkk-related nucleic acid. Another embodiment of the inventionprovides an isolated nucleic acid molecules in a form suitable forexpression of mRNA. In another embodiment, the isolated nucleic acidmolecules are in a form suitable for expression of protein. In yetanother embodiment, the isolated nucleic acid molecules are free fromvector sequences.

Another aspect of the invention provides a vector comprising a Dkknucleic acid molecule or Dkk-related nucleic acid molecule. In certainembodiments, the vector is a recombinant expression vector. In anotherembodiment, the invention provides a host cell containing a vector ofthe invention. The invention also provides a method for producing a Dkkprotein or Dkk-related protein by culturing in a suitable medium, a hostcell of the invention containing a recombinant expression vector suchthat a Dkk protein or Dkk-related protein is produced.

Another aspect of this invention features isolated or recombinant Dkkproteins and polypeptides or Dkk-related proteins and polypeptides. Inone embodiment, an isolated Dkk protein has a signal sequence and acysteine-rich region which comprises two cysteine-rich domains, and issecreted. In another embodiment, an isolated Dkk protein has an aminoacid sequence sufficiently homologous to the amino acid sequence of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8. or SEQ ID NO:21. In a preferredembodiment, a Dkk protein has an amino acid sequence at least about 60%homologous to the amino acid sequence of SEQ ID NO:2. In anotherpreferred embodiment, a Dkk protein has an amino acid sequence at leastabout 60% homologous to the amino acid sequence of SEQ ID NO:5. Inanother preferred embodiment, a Dkk protein has an amino acid sequenceat least about 60% homologous to the amino acid sequence of SEQ ID NO:8.In another preferred embodiment, a Dkk protein has an amino acidsequence at least about 75% homologous to the amino acid sequence of SEQID NO:8. In another preferred embodiment, a Dkk protein has an aminoacid sequence at least about 65% homologous to the amino acid sequenceof SEQ ID NO:21. In another embodiment. a Dkk protein has the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:21. Inanother preferred embodiment, a protein of the present invention has anamino acid sequence at least about 60% homologous to the amino acidsequence of SEQ ID NO:14. In another embodiment, a protein has the aminoacid sequence of SEQ ID NO:14.

Another embodiment of the invention features an isolated Dkk proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequenceat least about 60% homologous to a nucleotide sequence of SEQ ID NO:1,or a complement thereof. Another embodiment of the invention features anisolated Dkk protein which is encoded by a nucleic acid molecule havinga nucleotide sequence at least about 80% homologous to a nucleotidesequence of SEQ ID NO:4, or a complement thereof. Another embodiment ofthe invention features an isolated Dkk protein which is encoded by anucleic acid molecule having a nucleotide sequence at least about 60%homologous to a nucleotide sequence of SEQ ID NO:7, or a complementthereof. Another embodiment of the invention features an isolated Dkkprotein which is encoded by a nucleic acid molecule having a nucleotidesequence at least about 85% homologous to a nucleotide sequence of SEQID NO:7, or a complement thereof. Another embodiment of the inventionfeatures an isolated Dkk protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 70% homologous to anucleotide sequence of SEQ ID NO:20. or a complement thereof. Anotherembodiment of the invention features an isolated protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 90% homologous to a nucleotide sequence of SEQ ID NO:13, or acomplement thereof. This invention further features an isolated proteinwhich is encoded by a nucleic acid molecule having a nucleotide sequencewhich hybridizes under stringent hybridization conditions to a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1. SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, or a complement thereof.

The proteins of the present invention, or biologically active portionsthereof, can be operatively linked to a non-Dkk polypeptide ornon-Dkk-related polypeptide to form fusion proteins. The inventionfurther features antibodies that specifically bind Dkk or Dkk-relatedproteins, such as monoclonal or polyclonal antibodies. In addition, theproteins or biologically active portions thereof can be incorporatedinto pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingDkk expression (or the expression of a Dkk-related molecule) in abiological sample by contacting the biological sample with an agentcapable of detecting a nucleic acid molecule, protein or polypeptide ofthe present invention such that the presence of a Dkk (of Dkk-related)nucleic acid molecule, protein or polypeptide is detected in thebiological sample.

In another aspect. the present invention provides a method for detectingthe presence of a Dkk activity (or Dkk-related activity) in a biologicalsample by contacting the biological sample with an agent capable ofdetecting an indicator of Dkk activity (or Dkk-related activity) suchthat the presence of the activity is detected in the biological sample.

In another aspect, the invention provides a method for modulating Dkkactivity (or Dkk-related activity) comprising contacting the cell withan agent that modulates the activity such that the activity in the cellis modulated. In one embodiment, the agent inhibits Dkk activity (orDkk-related activity). In another embodiment, the agent stimulates Dkkactivity (or Dkk-related activity). In one embodiment, the agent is anantibody that specifically binds to a Dkk (or Dkk-related) protein. Inanother embodiment, the agent modulates expression of a protein (e.g., aDkk or a Dkk-related protein) by modulating transcription of a gene ortranslation of a mRNA of the present invention. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of a mRNA or gene of thepresent invention.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant expressionor activity of a protein or nucleic acid of the invention byadministering to the subject an agent which is a modulator of Dkk or aDkk-related molecule. In one embodiment, the modulator is a Dkk orDkk-related protein. In another embodiment the modulator is a Dkk orDkk-related nucleic acid molecule. In yet another embodiment, themodulator is an antibody peptide, peptidomimetic, or other smallmolecule. In a preferred embodiment, the disorder characterized byaberrant protein or nucleic acid expression is a developmental,differentiative, or proliferative disorder.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aDkk or Dkk-related protein; (ii) misregulation of said gene; and (iii)aberrant post-translational modification of a Dkk or Dkk-relatedprotein, wherein a wild-type form of said gene encodes an protein with aDkk or Dkk-related activity.

In another aspect the invention provides a method for identifying acompound that binds to or modulates the activity of a Dkk or Dkk-relatedprotein, by providing a indicator composition comprising a Dkk orDkk-related protein having a biological activity, contacting theindicator composition with a test compound, and determining the effectof the test compound on the activity in the indicator composition toidentify a compound that modulates the activity of a Dkk or Dkk-relatedprotein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1, 1A-2, 1B-1, 1B-2 depicts the cDNA sequence and predictedamino acid sequence of human Dkk-3. The nucleotide sequence correspondsto nucleic acids 1 to 2479 of SEQ ID NO:1. The amino acid sequencecorresponds to amino acids 1 to 350 of SEQ ID NO:2.

FIG. 2A-2B depicts the cDNA sequence and predicted amino acid sequenceof human Dkk-4. The nucleotide sequence corresponds to nucleic acids 1to 848 of SEQ ID NO:4. The amino acid sequence corresponds to aminoacids 1 to 224 of SEQ ID NO:5.

FIG. 3A-3B depicts the cDNA sequence and predicted amino acid sequenceof human Dkk-1. The nucleotide sequence corresponds to nucleic acids 1to 1536 of SEQ ID NO:7. The amino acid sequence corresponds to aminoacids 1 to 266 of SEQ ID NO:8.

FIG. 4A-1, 4A-2, 4A-3, 4B-1, 4B-2 depicts the cDNA sequence andpredicted amino acid sequence of full-length human Dkk-2. The nucleotidesequence corresponds to nucleic acids 1 to 3687 of SEQ ID NO:20. Theamino acid sequence corresponds to amino acids 1 to 259 of SEQ ID NO:21.

FIG. 5A-1, 5A-2, 5B-1, 5B-2 depicts the cDNA sequence and predictedamino acid sequence of murine Dkk-3. The nucleotide sequence correspondsto nucleic acids 1 to 2381 of SEQ ID NO:16. The amino acid sequencecorresponds to amino acids 1 to 349 of SEQ ID NO:17.

FIG. 6A-6C depicts a multiple sequence alignment of the amino acidsequences of hDkk-1 (corresponding the SEQ ID NO:8), mDkk-1(corresponding to SEQ ID NO:36 and having Accession No. AF030434),Xenopus Dkk-1 (“xDkk-1”) (corresponding to SEQ ID NO:37 and havingAccession No. AF030433), hDkk-2 (corresponding to SEQ ID NO:21), hDkk-3(corresponding to SEQ ID NO:2), mDkk-3 (corresponding to SEQ ID NO:17),chicken Dkk-3 (“cDkk-3”) (corresponding to SEQ ID NO:38 and havingAccession No. D26311), and hDkk-4 (corresponding to SEQ ID NO:5). Thealignment was performed using the ClustalW algorithm as implemented inthe GCG program PILEUP. The alignment provides information regarding therelationship between the Dkk proteins of the instant invention.Predicted signal peptides are underlined, N-glycosylation sites areindicated by a thick bar, CRD-1 by an open box. CRD-2 by a shaded box.The proteolytic cleavage site within hDkk4 is indicated by an arrow.

FIG. 7A-7B depicts the cDNA sequence and predicted amino acid sequenceof human Soggy. The nucleotide sequence corresponds to nucleic acids 1to 928 of SEQ 5 ID NO:13. The amino acid sequence corresponds to aminoacids 1 to 242 of SEQ ID NO:14.

FIG. 8A-8B depicts the cDNA sequence and predicted amino acid sequenceof murine Soggy-1. The nucleotide sequence corresponds to nucleic acids1 to 835 of SEQ ID NO:26. The amino acid sequence corresponds to aminoacids 1 to 230 of SEQ ID 10 NO:27.

FIG. 9 is a schematic diagram illustrating the Dkk and Dkk-relatedproteins of the instant invention. The figure depicts the structuraldomains of the human Dkks and Soggy. Signal peptides are indicated bydarkened boxes. The cysteine-rich domains of a Dkk cysteine-rich regionare depicted as CRD-1 and CRD-2. Branches indicate sites ofN-glycosylation.

FIG. 10A-10B depicts a multiple sequence alignment of hSoggy-1(corresponding to SEQ ID NO:14), murine Soggy-1 (corresponding to SEQ IDNO:27), hDkk-3 (corresponding to SEQ ID NO:2), and mDkk-3 (correspondingto SEQ ID NO:17). The alignment was generated as described in the legendto FIG. 6. The alignment provides details regarding the relationshipbetween the Dkk-3 and Soggy-1 proteins of the instant invention.Predicted signal peptides are underlined, N-glycosylation sites areindicated by a thick bar. CRD-1 and CRD-2 within Dkk-3 are indicated forreference by open and shaded boxes.

FIG. 11 depicts a multiple sequence alignment of the carboxy-terminalcysteine-rich domains of hDkk-1 (corresponding to amino acid residues181-266 of SEQ ID NO:8), hDkk-2 (corresponding to amino acid residues179-263 of SEQ ID NO:21), hDkk-3 (corresponding to amino acid residues200-292 of SEQ ID NO:2), hDkk-4 (corresponding to amino acid residues137-224 of SEQ ID NO:5) with human colipase (corresponding to SEQ IDNO:25 and having accession No. J02883). The carboxy-terminalcysteine-rich domains of the Dkk proteins are indicated by an open box.The alignment was generated using PILEUP (gap penalties of 12 foropening and 12 for extending). A minor adjustment was necessary sincePILEUP inserts a single gap in hDkk-1 and hDkk-2 between Gly56 andSer57, even with a gap opening penalty of 15. The conserved residues areindicated. The disulfide-bonding pattern typical for the colipase familyand predicted for the Dkk family is indicated below the alignment.

FIG. 12 is a schematic diagram depicting the relationship between thehDkk-3 nucleotide sequence (corresponding to SEQ ID NO:1) and those ofRIG and RIG-like 7-1 (Accession Nos. U32331 and AF034208, respectively).Thick bars indicate regions of sequence identity between hDkk-3 and RIGor RIG-like 7-1 mRNAs. As between RIG and hDkk-3, there exists a shortregion of identity within the 3′ untranslated regions of the mRNAs whenthe mRNAs are aligned in reverse orientation. As between hDkk-3 andRIG-like 7-1, there exists a longer region of identity. however,RIG-like 7-1 lacks a signal sequence and, accordingly, is not predictedto be secreted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel molecules,referred to herein as Dkk protein and nucleic acid molecules, whichcomprise a family of molecules having certain conserved structural andfunctional features. The term “family” when referring to the protein andnucleic acid molecules of the invention is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally-occurring and can be fromeither the same or different species. For example, a family can containa first protein of human origin, as well as other, distinct proteins ofhuman origin or alternatively, can contain homologues of non-humanorigin. Members of a family may also have common functionalcharacteristics.

In one embodiment, a Dkk family member is identified based on thepresence of at least one “cysteine-rich domain” in the protein moleculeor corresponding amino acid sequence. As defined herein, a“cysteine-rich domain” refers to a portion of a Dkk protein (e.g.,hDkk-3) which is rich in cysteine residues. In a preferred embodiment, a“cysteine-rich domain” is a protein domain having an amino acid sequenceof about 45-85 amino acids of which preferably 10 amino acids arecysteine residues located at the same relative amino acid position asthe cysteine residues in human Dkk-3 having SEQ ID NO:2 (e.g., aminoacid residues 147-195 of SEQ ID NO:2). In another embodiment, a“cysteine-rich domain” has 30-100 amino acids, preferably about 35-95amino acids, more preferably about 40-90 amino acids, more preferablyabout 50-80 amino acids, even more preferably about 51-75, 60-70, or 65amino acids, of which at least about 3-20, preferably about 5-15, ormore preferably about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acids are cysteine residues.

A preferred Dkk protein of the present invention has a firstcysteine-rich domain (“CRD-1”) referred to herein as an “amino-terminalcysteine-rich domain” or “N-terminal cysteine-rich domain” and a secondcysteine-rich domain (“CRD-2”), referred to herein as a“carboxy-terminal cysteine-rich domain” or “C-terminal cysteine-richdomain”. As defined herein, an “amino-terminal cysteine-rich domain” isa protein domain having an amino acid sequence of about 45-55 aminoacids of which preferably 10 amino acids are cysteine residues locatedat the same relative position as the cysteine residues in anamino-terminal cysteine-rich domain of human Dkk-3 having SEQ ID NO:2(e.g., amino acid residues 147-195 of SEQ ID NO:2). In anotherembodiment, an “amino-terminal cysteine-rich domain” has 30-70,preferably 35-65, more preferably about 40-60, and even more preferablyabout 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acids, of which atleast about 3-20, preferably about 5-15, or more preferably about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids arecysteine residues. In a preferred embodiment, an amino-terminalcysteine-rich domain has the following consensus sequence:C-X(2)-D-X(2)-C-X(5)-C-X(8-13)-C-X(2)-C-X(6)-C-X(5)-C-C-X(4)-C-X(4)-C(SEQID NO:23). The consensus sequences described herein are describedaccording to standard Prosite Signature designation (e.g., all aminoacids are indicated according to their universal single letterdesignation; X designates any amino acid; X(n) designates any n aminoacids, e.g., X (2) designates any 2 amino acids; and [LIVM] indicatesany one of the amino acids appearing within the brackets, e.g., any oneof L, I, V, or M, in the alternative, any one of Leu, Ile, Val, or Met.)

As defined herein, a “carboxy-terminal cysteine-rich domain” is aprotein domain having an amino acid sequence of about 80-85 amino acidsof which preferably 10 amino acids are cysteine residues located at thesame relative position as the cysteine residues in a carboxy-terminalcysteine-rich domain of human Dkk-3 having SEQ ID NO:2 (e.g., amino acidresidues 201-284 of SEQ ID NO:2). In another embodiment, a“carboxy-terminal cysteine-rich domain” has 65-100, preferably 70-95.more preferably about 75-90, and even more preferably about 81, 82, 83,or 84 amino acids, of which at least about 3-20, preferably about 5-15,or more preferably about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acids are cysteine residues. In a preferred embodiment,a carboxy-terminal cysteine-rich domain has the following consensussequence:C-X(4)-D-C-X(2)-G-X-C-C-X(8-10)-C-X-P-X(4)-G-X(2)-C-X(16-24)-C-X-C-X(2)-P-X(4)-G-X(2)-C-X(16-24)-C-X-C-X(2)-G-L-X-C-X(10-17)-C(SEQ ID NO:24).

A preferred protein of the present invention is a hDkk-3 protein (humanDkk-3) containing an amino-terminal cysteine-rich domain including aboutamino acids 147-195 of SEQ ID NO:2, having 10 cysteine residues. and acarboxy-terminal cysteine-rich domain including about amino acids201-284 of SEQ ID NO:2, having 10 cysteine residues (the positions ofthe cysteine residues are depicted in FIG. 6A-C). In another embodiment,a hDkk-4 (human Dkk-4) protein contains an amino-terminal cysteine-richdomain including about amino acids 41-90 of SEQ ID NO:5, having 10cysteine residues, and a carboxy-terminal cysteine-rich domain includingabout amino acids 138-218 of SEQ ID NO:5, having 10 cysteine residues(the positions of the cysteine residues are depicted in FIG. 6A-C). Inanother embodiment. a hDkk-1 protein (human Dkk-1) contains anamino-terminal cysteine-rich domain including about amino acids 85-138of SEQ ID NO:8, having 10 cysteine residues, and a carboxy-terminalcysteine-rich domain including about amino acids 182-263 of SEQ ID NO:8,having 10 cysteine residues (the positions of the cysteine residues aredepicted in FIG. 6A-C). In another embodiment, a hDkk-2 protein (humanDkk-2) contains an amino-terminal cysteine-rich domain including aboutamino acids 78-127 of SEQ ID NO:21, having 10 cysteine residues, and acarboxy-terminal cysteine-rich domain including about amino acids176-256 of SEQ ID NO:21, having 10 cysteine residues (the positions ofthe cysteine residues are depicted in FIG. 6A-C).

Alignment of the human Dkk proteins with human colipase (havingAccession No. J02883) indicates that the carboxy-terminal cysteine-richdomains of the human Dkk proteins have a pattern of cysteines typical ofcolipase (FIG. 11 and Avarind and Koonin, supra). Within colipase, thesecysteine residues are involved in disulfide bonding which gives rise toa structure termed the “colipase fold”. The “colipase fold” is typicalof a range of small proteins which are involved in protein-proteininteractions including, but not limited to the colipases, snake andscorpion toxins and protease inhibitors (Hubbard et al. (1997) NucleicAcids Res. 25:236-239. These proteins have a series of short 3 strandswith large connecting loops, which are held together by disulfide bonds.The disulfide-bonding pattern typical for colipase and predicted for theDkk family is indicated below the alignment of FIG. 11. Conservedhydrophobic residues between the Dkks and human colipase suggest thatthe Dkks, like the colipases, interact with lipids (e.g., Leu51 of humancolipase, SEQ ID NO:25 which corresponds to Leu271 of hDkk-3 (SEQ IDNO:2); Leu200 of hDkk-4 (SEQ ID NO:5): Leu243 of hDkk-1 (SEQ ID NO:8);and Leu237 of hDkk-2 (SEQ ID NO:21). The carboxy terminal cysteine-richdomain of the Dick family, may function in the membrane association ofDkk, which in turn may be required for the inhibition of Wnt secretionor Wnt:7 transmembrane receptor interaction. In addition, inhibition ofWnt function by the Dkk family may be closely associated with the cellmembrane and the carboxy-terminal cysteine-rich domain of the Dkk familymay mediate this association. Furthermore, the amino-terminalcysteine-rich domain of the Dkk family may directly interact with Wnt orits receptor. Accordingly, a preferred Dkk protein of the presentinvention comprises a carboxy-terminal cysteine-rich domain. In oneembodiment, a Dkk protein comprising a carboxy-terminal cysteine-richdomain lacks the amino-terminal cysteine-rich domain.

In a preferred embodiment, the cysteine residues of a cysteine-richdomain are located at the same relative amino acid position as thecysteine residues in human Dkk-3 having SEQ ID NO:2. In anotherpreferred embodiment, the cysteine residues of a cysteine-rich domainare located at the same relative position as the cysteine residues in acysteine-rich domain of human Dkh-3 having SEQ ID NO:2. For example. asshown in FIG. 6A-C, human Dkk-4 has at least about 10 cysteine residueslocated at the same relative amino acid position as the cysteineresidues in human Dkk-3 having SEQ ID NO:2 (e.g., cys151 in Dkk-4, SEQID NO:5. is located at the same relative amino acid position as cys214in Dkk-3, SEQ ID NO:2; cys156 in Dkk-4, SEQ ID NO:5, is located at thesame relative amino acid position as cys219 in Dkk-3, SEQ ID NO:2; andcys157 in Dkk-4, SEQ ID NO:5, is located at the same relative amino acidposition as cys220 in Dkk-3. SEQ ID NO:2). Similarly, as shown in FIG.6A-C, Dkk-1 has at least about 10 cysteine residues located at the samerelative amino acid position as the cysteine residues in human Dkk-3having SEQ ID NO:2. As also shown in FIG. 6A-C. Dkk-2 has at least about10 cysteine residues located at the same relative amino acid position asthe cysteine residues in human Dkk-3 having SEQ ID NO:2. Table I setsforth at least 20 cysteine residues in each of hDkk-4, hDkk-1, andhDkk-2 which are located in the same relative position as 20 cysteineresidues in hDkk-3.

TABLE I as position as position as position as position cysteine inhDkk-3 in hDkk-4 in hDkk-1 in hDkk-2 1 147 41 85 78 2 153 47 91 84 3 15953 97 90 4 168 63 111 100 5 171 66 114 103 6 178 73 121 110 7 184 79 127116 8 185 80 128 117 9 190 85 133 122 10 195 90 138 127 11 208 145 189183 12 214 151 195 189 13 219 156 200 194 14 220 157 201 195 15 231 166210 204 16 241 176 220 214 17 265 194 237 231 18 267 196 239 233 19 273202 245 239 20 284 218 263 256

The first 10 rows of Table I contain 10 cysteine residues that areincluded within the first, or amino-terminal, cysteine-rich domain ofeach of hDkks-3, -4, -1, and -2. The last 10 rows of Table I contain 10cysteine residues that are included within the second, orcarboxy-terminal, cysteine-rich domain of each of hDkks-3, -4, -1, and-2.

Preferred Dkk proteins have more than one cysteine-rich domain, morepreferably have at least two cysteine-rich domains and, thus, have acysteine-rich region. As used herein, the term “cysteine-rich region”refers to a protein domain which includes at least two cysteine-richdomains and has an amino acid sequence of about 120-200 amino acidresidues of which at least about 20 of the amino acids are cysteineresidues. In another embodiment, a “cysteine-rich region” has preferablyabout 140-180 amino acid residues, and even more preferably at leastabout 135-175 amino acids of which at least about 10-30, preferablyabout 15-20, and more preferably about 16, 17, 18, or 19 of the aminoacids are cysteine residues. In a preferred embodiment, a cysteine-richregion is located in the C-terminal region of a Dkk protein. Forexample, in one embodiment, a hDkk-3 protein contains a cysteine richregion containing about amino acids 147-284 of SEQ ID NO:2, having 20cysteine residues at the positions indicated in FIG. 6A-C. In anotherembodiment, a hDkk-4 protein contains a cysteine rich region containingabout amino acids 41-218 of SEQ ID NO:5, having 20 cysteine residues atthe positions indicated in FIG. 6A-C. In another embodiment, a hDkk-1protein contains a cysteine rich region containing about amino acids85-263 of SEQ ID NO:8, having 20 cysteine residues at the positionsindicated in FIG. 6A-C. In another embodiment, a hDkk-2 protein containsa cysteine rich region containing about amino acids 78-256 of SEQ IDNO:21, having 20 cysteine residues at the positions indicated in FIG.6A-C.

In another embodiment, in addition to cysteine-rich domains, thecysteine-rich region contains a spacer region which separates the firstand second cysteine-rich domains. As used herein, the “spacer region”refers to amino acid residues which are located between the first andsecond cysteine-rich domains of a cysteine-rich region and includesamino acid residues located C-terminal to the first cysteine-rich domainand N-terminal to the second cysteine-rich domain. As defined herein, a“spacer region” refers to a protein domain of about 5-70 amino acids,preferably about 10-65 amino acids, more preferably about 15-60 aminoacids, even more preferably about 20-55 amino acids, and even morepreferably about 25-50, 30-45 or 35-40 amino acids. For example, hDkk-3protein contains a spacer region of about amino acids 196-200 of SEQ IDNO:2; hDkk-4 protein contains a spacer region of about amino acids91-137 of SEQ ID NO:5; hDkk-1 protein contains a spacer region of aboutamino acids 139-181 of SEQ ID NO:8; and hDkk-2 protein contains a spacerregion of about amino acids 128-175 of SEQ ID NO:21. The spacer regionsof hDkk-1, hDkk-2 and hDkk-4 are remarkably conserved in length (e.g.,the spacer region of hDkk-1 consists of 43 amino acid residues, thespacer region of hDkk-2 consists of 48 amino acid residues and thespacer region of hDkk-4 consists of 47 amino acid residues, suggestingthat the close proximity of CRD-1 and CRD-2 is important in Dkkfunction. Accordingly, in one embodiment, the spacer region functions tospacially restrict the separation of CRD-1 from CRD-2.

In another embodiment of the invention, the Dkk protein has at least onecysteine-rich domain, preferably a cysteine-rich region, and a signalsequence. As used herein, a “signal sequence” refers to a peptidecontaining about 18-24 amino acids which occurs at the N-terminus ofsecretory and integral membrane proteins and which contains at leastabout 40-70% hydrophobic amino acid residues (e.g., alanine, valine,leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline).In another embodiment, a signal sequence contains at least about 8-34,9-33, 10-32, 11-31, 12-30, 13-29, 14-28 amino acid residues, preferablyabout 15-27 amino acid residues, more preferably about 16-26 amino acidresidues, more preferably about 17-25 amino acid residues, and morepreferably about 18-24, 19-23, 20-22, or 21 amino acid residues, and hasat least about 50-65%, and more preferably about 55-60% hydrophobicamino acid residues (e.g., alanine, valine, leucine, isoleucine,phenylalanine, tyrosine, tryptophan, or proline). Such a “signalsequence”, also referred to in the art as a “signal peptide”, serves todirect a protein containing such a sequence to a lipid bilayer. Forexample, in one embodiment, a hDkk-3 protein contains a signal sequenceof about amino acids 1-23 of SEQ ID NO:2. In another embodiment, ahDkk-4 protein contains a signal sequence of about amino acids 1-19 ofSEQ ID NO:5. In another embodiment, a hDkk-1 protein contains a signalsequence of about amino acids 1-20 of SEQ ID NO:8. In anotherembodiment, a hDkk-2 protein contains a signal sequence of about aminoacids 1-33 of SEQ ID NO:21. A preferred Dkk protein of the presentinvention is a human protein (e.g., encoded by a nucleotide sequencecorresponding to a naturally-occurring human gene).

Accordingly, one embodiment of the invention features a Dkk proteinhaving at least one cysteine-rich domain, preferably at least onecysteine-rich region. Another embodiment features a Dkk protein havingat least one cysteine-rich region, wherein the cysteine-rich regionincludes at least one cysteine-rich domain. Another embodiment featuresa Dkk protein having at least one cysteine-rich region. wherein thecysteine-rich region includes at least two cysteine-rich domains.Another embodiment features a protein or domain within a protein having20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% homology to a cysteine-richdomain of a Dkk protein of the invention (e.g., hDkk-3, hDkk-4, hDkk-1,or hDkk-2).

Yet another embodiment of the invention features a Dkk protein having atleast one cysteine-rich domain, preferably at least one cysteine-richregion and a signal peptide. Another embodiment features a Dkk proteinhaving at least one cysteine-rich domain, preferably at least onecysteine-rich region and a signal peptide, wherein the cysteine-richregion includes at least two cysteine-rich domains. Another embodimentfeatures a Dkk protein having at least one cysteine-rich domain,preferably at least one cysteine-rich region and a signal peptide,wherein the cysteine-rich region includes at least two cysteine-richdomains and a spacer.

Yet another aspect of the invention features Dkk proteins having domainsand/or regions which are conserved among a subset of Dkk proteins butare not necessarily conserved among all Dkk family members. In oneembodiment, a Dkk protein (e.g., Dkk-3) has an “extended N-terminalregion” which is extended in length as compared to, for example, the“N-terminal regions” of other Dkk family members (e.g., Dkk-4, Dkk-1,and Dkk-2). As defined herein, an “N-terminal region” of a Dkk proteinsconsists of amino acid residues found between the signal peptide andCRD-1 of a Dkk protein. Preferably, the first amino acid residue of anN-terminal region of Dkk is the first residue of a mature Dkk proteinand the last residue of an N-terminal region of Dkk is the residuepreceeding the first cysteine residue of CRD-1. In a preferredembodiment, an N-terminal region is about 1-20 amino acid residues inlength, preferably about 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90, 91-100, 101-110, 111-120, 121-130, 131-140, 141-150, 151-160 ormore amino acid residues in length. In contrast, an “extended N-terminalregion” is at least about 71-80, 81-90, 91-100, 101-110, 111-120,121-130, 131-140, 141-150, 151-160 or more amino acid residues inlength. For example, in one embodiment, a hDkk-4 protein includes an“N-terminal region” of about amino acids 20-40 of SEQ ID NO:5 (21 aminoacid residues in length). In another embodiment, a hDkk-1 proteinincludes an N-terminal region of about amino acids 21-84 of SEQ ID NO:8(64 amino acid residues in length). In another embodiment, a hDkk-2protein includes an “N-terminal region” of about amino acids 34-77 ofSEQ ID NO:21 (44 amino acid residues in length). In another embodiment,a hDkk-3 protein has an “extended N-terminal region” of about aminoacids 23-146 of SEQ ID NO:2 (124 amino acid residues in length).

In another embodiment, a Dkk protein (e.g., Dkk-3) has an “acidicC-terminal region” which includes amino acid residues found C-terminalto CRD-2 of a Dkk protein. Preferably, the first amino acid residue ofan acidic C-terminal region is the residue following the last cysteineof CRD-2 and the last residue of an acidic C-terminal region is the lastresidue of a Dkk protein. In a preferred embodiment. an acidicC-terminal region is about 65-66 amino acid residues in length and hasabout 27-25% acidic amino acid residues (e.g., glutamic acid or asparticacid). In another preferred embodiment. an acidic C-terminal region isabout 55-80 amino acid residues in length. preferably about 60-75 aminoacid residues in length. and more preferably about 64-70 amino acidresidues in length and has about 21-35% acidic amino acid residues,preferably about 23-33% acidic amino acid residues, and more preferablyabout 25-31% acidic amino acid residues. Preferably, an acidicC-terminal region is involved in protein-protein interactions. Forexample, in one embodiment, a hDkk-3 protein has an acidic C-terminalregion from about amino acids 285-350 of SEQ ID NO:2.

Preferred Dkk molecules of the present invention have an amino acidsequence sufficiently homologous to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:21. As used herein, theterm “sufficiently homologous” refers to a first amino acid ornucleotide sequence which contains a sufficient or minimum number ofidentical or equivalent (e.g., an amino acid residue which has a similarside chain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences share common structural domains and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains have at least about 40% homology,preferably 50% homology, more preferably 60%-70% homology across theamino acid sequences of the domains and contain at least one, preferablytwo, more preferably three, and even more preferably four, five or sixstructural domains, are defined herein as sufficiently homologous.Furthermore, amino acid or nucleotide sequences which share at least40%, preferably 50%, more preferably 60, 70, or 80% homology and share acommon functional activity are defined herein as sufficientlyhomologous.

As used interchangeably herein, a “Dkk activity”, “biological activityof Dkk” or “functional activity of Dkk”, refers to an activity exertedby a Dkk protein, polypeptide or nucleic acid molecule (e.g., anactivity on a Dkk responsive cell) as determined in vivo, or in vitro,according to standard techniques. In one embodiment, a Dkk activity is adirect activity, such as an association with a Dkk-target molecule. Asused herein, a “target molecule” is a molecule with which a Dkk proteinbinds or interacts in nature, such that Dkk-mediated function isachieved. A Dkk target molecule can be a non-Dkk molecule or a Dkkprotein or polypeptide of the present invention. In an exemplaryembodiment, a Dkk target molecule is a membrane-bound protein (e.g., acell-surface receptor or “Dkk receptor”) or a modified form of such aprotein which has been altered such that the protein is soluble (e.g.,recombinantly produced such that the protein does not express amembrane-binding domain). In another embodiment, a Dkk target is asecond soluble protein molecule (e.g., a “Dkk binding partner” or “Dkksubstrate”). In such an exemplary embodiment. a Dkk binding partner canbe a second soluble non-Dkk protein or a second Dkk protein molecule ofthe present invention. Alternatively, a Dkk activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the Dkk protein with a second protein (e.g., a Dkk receptor). As usedherein, the term “Dkk receptor” refers to a protein or protein complex,to which a Dkk protein, e.g., human Dkk can bind. A receptor can be acell surface receptor, e.g., a peptide. growth factor, or nuclearhormone receptor. Dkk receptors can be isolated by methods known in theart and further described herein. Interaction of a Dkk protein with aDkk receptor can result in transduction of a signal from the cellsurface to the nucleus. The signal transduced can be, an increase inintracellular calcium, an increase in phosphatidylinositol or othermolecule, and can result in, e.g., in phosphorylation of specificproteins, a modulation of gene transcription and any of the otherbiological activities set forth herein.

In a preferred embodiment, a Dkk activity is at least one or more of thefollowing activities: (i) interaction of a Dkk protein with and/orbinding to a second molecule. (e.g., a protein, such as a Dkk receptor,a soluble form of a Dkk receptor, a receptor for a member of the wntfamily of signaling proteins, or a non-Dkk signaling molecule, forexample, a lipid included in a cell membrane); (ii) interaction of a Dkkprotein with an intracellular protein via a membrane-bound Dkk receptor;(iii) complex formation between a soluble Dkk protein and a secondsoluble Dkk binding partner (e.g., a non-Dkk protein molecule or asecond Dkk protein molecule); (iv) interaction with other extracellularproteins (e.g., regulation of wnt-dependent cellular adhesion toextracellular matrix components); (v) binding to and eliminating anundesirable molecule (e.g., a detoxifying activity or defense function);and/or (vi) an enzymatic activity. In yet another preferred embodiment,a Dkk activity is at least one or more of the following activities: (1)modulation of cellular signal transduction, either in vitro or in vivo(e.g., modulation, e.g., antagonism, of the activity of members of thewnt family of secreted proteins or suppression of wnt-dependent signaltransduction, for example suppression of Wnt-2b, Wnt3 and/orWnt8-dependent signal transduction by hDkk-1 and/or hDkk-4); (2)regulation of communication between cells (e.g., regulation ofwnt-dependent cell-cell interactions); (3) regulation of expression ofgenes whose expression is modulated by binding of Dkk (e.g., hDkk-3) toa receptor; (4) regulation of gene transcription in a cell involved indevelopment or differentiation. either in vitro or in vivo (e.g.,induction of cellular differentiation); (5) regulation of genetranscription in a cell involved in development or differentiation,wherein at least one gene encodes a differentiation-specific protein;(6) regulation of gene transcription in a cell involved in developmentor differentiation, wherein at least one gene encodes a second secretedprotein; (7) regulation of gene transcription in a cell involved indevelopment or differentiation. wherein at least one gene encodes asignal transduction molecule; (8) regulation of cellular proliferation,either in vitro or in vivo (e.g. induction of cellular proliferation orinhibition of proliferation as in the case of suppression oftumorigenesis (e.g., suppression of glial cell tumor growth, forexample, glioblastoma growth)); (9) formation and maintenance of orderedspatial arrangements of differentiated tissues in vertebrates, bothadult and embryonic (e.g., induction of head formation during vertebratedevelopment or maintenance of hematopoietic progenitor cells); (10)modulation of cell death, such as stimulation of cell survival; (11)regulating cell migration; and/or (12) immune modulation.

As referred to herein, “differentiation-specific proteins” includeproteins involved in the transition of a cell from the undifferentiatedto the differentiated phenotype. For example, such proteins can bedifferentiation specific structural proteins or differentiation-specifictranscription factors. Such differentiation-specific proteins aregenerally expressed at higher levels in cells which are making thetransition from the undifferentiated to the differentiated phenotype(e.g., during embryonic development or during regeneration of maturetissue in the adult animal), or are expressed at higher levels infully-differentiated or terminally-differentiated cells as compared totheir undifferentiated counterparts. Also, as referred to herein,“differentiation-specific genes” include nucleic acid molecules whichencode differentiation-specific proteins.

Accordingly, another embodiment of the invention features isolated Dkkproteins and polypeptides having a Dkk activity, Preferred Dkk proteinshave at least one cysteine-rich region and a Dkk activity. In anotherpreferred embodiment, the Dkk protein has at least one cysteine-richregion, wherein the cysteine-rich region comprises at least onecysteine-rich domain, and a Dkk activity. In another preferredembodiment. the Dkk protein has at least one cysteine-rich region,wherein the cysteine-rich region comprises at least two cysteine-richdomains, and a Dkk activity. In yet another preferred embodiment, a Dkkprotein further comprises a signal sequence. In still another preferredembodiment. a Dkk protein has a cysteine-rich region, a Dkk activity,and an amino acid sequence sufficiently homologous to an amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:21.

A preferred Dkk fragment comprises a carboxy-terminal cysteine-richdomain. In one embodiment, a Dkk fragment comprises a carboxy-terminalcysteine-rich domain and retains a biological activity of a Dkk protein.In yet another embodiment, a Dkk fragment lacks an amino-terminalcysteine-rich domain.

The human Dkk-3 cDNA, which is approximately 2479 nucleotides in length,encodes a protein which is approximately 350 amino acid residues inlength. The human Dkk-3 protein contains an N-terminal signal sequenceand a cysteine-rich region comprising two cysteine-rich domains. A Dkkcysteine-rich region can be found at least, for example, from aboutamino acids 147-284 of SEQ ID NO:2. The hDkk-3 cysteine-rich regioncomprises an amino-terminal cysteine-rich domain from about amino acids147-195 of SEQ ID NO:2 and a carboxy-terminal cysteine-rich domain fromabout amino acids 201-284 of SEQ ID NO:2. The human Dkk-3 protein is asecreted protein which further contains a signal sequence at about aminoacids 1-21, 1-22, 1-23, or 1-24 of SEQ ID NO:2. Accordingly, a maturehuman Dkk-3 protein begins at about amino acid residue 22, 23, 24, or 25of SEQ ID NO:2 and is about 329, 328, 327, or 326 amino acids in length.The prediction of such a signal peptide can be made, for example,utilizing the computer algorithm SIGNALP (Nielsen, et al., (1997)Protein Engineering 10:1-6).

The human Dkk-4 cDNA, which is approximately 848 nucleotides in length,encodes a protein which is approximately 224 amino acid residues inlength. The human Dkk-4 protein contains an N-terminal signal sequenceand a cysteine-rich region comprising two cysteine-rich domains. A Dkkcysteine-rich region can be found at least, for example, from aboutamino acids 41-218 of SEQ ID NO:5. The hDkk-4 cysteine-rich regioncomprises an amino-terminal cysteine-rich domain from about amino acids41-90 of SEQ ID NO:5 and a carboxy-terminal cysteine-rich domain fromabout amino acids 138-218 of SEQ ID NO:5. The human Dkk-4 protein is asecreted protein which further contains a signal sequence at about aminoacids 1-17, 1-18, 1-19, or 1-20 of SEQ ID NO:5. Accordingly, a maturehuman Dkk-4 protein begins at about amino acid residue 18, 19, 20, or 21of SEQ ID NO:5 and is about 207, 206, 205, or 204 amino acids in length.A preferred fragment of hDkk-4 comprises amino acid residues 134-224 ofSEQ ID NO:5. In another embodiment. a preferred fragment of hDkk-4consists of amino acid residues 134-224 of SEQ ID NO:5.

The human Dkk-1 cDNA, which is approximately 1536 nucleotides in length,encodes a protein which is approximately 266 amino acid residues inlength. The human Dkk-1 protein contains an N-terminal signal sequenceand a cysteine-rich region comprising two cysteine-rich domains. A Dkkcysteine-rich region can be found at least, for example, from aboutamino acids 85-263 of SEQ ID NO:8. The hDkk-1 cysteine-rich regioncomprises an amino-terminal cysteine-rich domain from about amino acids85-138 of SEQ ID NO:8 and a carboxy-terminal cysteine-rich domain fromabout amino acids 182-263 of SEQ ID NO:8. The human Dkk-1 protein is asecreted protein which further contains a signal sequence at about aminoacids 1-18, 1-19, 1-20, or 1-21 of SEQ ID NO:8. Accordingly, a maturehuman Dkk-1 protein begins at about amino acid residue 19, 20, 21, or 32of SEQ ID NO:8 and is about 248, 247, 246, or 245 amino acids in length.

The human Dkk-2 cDNA, which is approximately 3687 nucleotides in length.encodes a protein which is approximately 259 amino acid residues inlength. The human Dkk-2 protein contains a cysteine-rich regioncomprising two cysteine-rich domains. A Dkk cysteine-rich region can befound at least, for example, from about amino acids 78-256 of SEQ IDNO:21. The hDkk-2 cysteine-rich region comprises an amino-terminalcysteine-rich domain from about amino acids 78-127 of SEQ ID NO:21 and acarboxy-terminal cysteine-rich domain from about amino acids 176-256 ofSEQ ID NO:21. The human Dkk-2 protein is a secreted protein whichfurther contains a signal sequence at about amino acids 1-31, 1-32,1-33, or 1-34 of SEQ ID NO:21. Accordingly, a mature human Dkk-2 proteinbegins at about amino acid residue 32, 33, 34, or 35 of SEQ ID NO:21 andis about 228, 227, ?26, or 225 amino acids in length.

Dkk proteins of the present invention can be used to identify additionalDkk-related proteins or family members. For example, a protein havinghomology to hDkk-3 was identified using the nucleotide sequence encodingthe N-terminal unique region of hDkk-3 to search a nucleotide sequencedatabase. A human cDNA clone (Accession No.: AA397836) was identifiedfrom the dBEST database as having homology to hDkk-3 and was fullysequenced. The encoded protein is referred to herein as human “Soggy-1”or “Dkk-like-N”. The nucleotide and predicted amino acid sequence ofhuman Soggy-1 are depicted in FIG. 7. The nucleotide sequence of humanSoggy-1 (SEQ ID NO:13) encodes a protein having 242 amino acids (SEQ IDNO:14). The nucleotide sequence of human Soggy-1 includes a 5′untranslated region containing nucleotides 1-74 of SEQ ID NO:13, acoding region containing nucleotides 75-800 of SEQ ID NO:13(corresponding to nucleotides 1-726 of SEQ ID NO:15), and a 3′untranslated region containing nucleotides 801-928 of SEQ ID NO:13. TheSoggy-1 protein (amino acid residues 32-132) has 25% identity to anN-terminal domain of human Dkk-3 (consisting of amino acid residues22-140) as determined by ALIGN, Myers and Miller, (1989) CABIOS,utilizing a PAM120 weight residue table, a gap length penalty of 12, anda gap penalty of 4.

Two murine cDNA clones were further identified from the database andfully sequenced. Combining the sequence information from these twoclones resulted in a full-length sequence for murine Soggy-1. Thenucleotide sequence and predicted amino acid sequence of murine Soggy-1are depicted in FIG. 8A-8B. The nucleotide sequence of murine Soggy-1(SEQ ID NO:26) encodes a protein having 230 amino acids (SEQ ID NO:27).The nucleotide sequence of murine Soggy-1 includes a 5′ untranslatedregion containing nucleotides 1-56 of SEQ ID NO:26, a coding regioncontaining nucleotides 57-746 of SEQ ID NO:26 (corresponding to SEQ IDNO:26), and a 3 untranslated region containing nucleotides 747-835 ofSEQ ID NO:26. Human and murine Soggy-1 proteins display 59% overallidentity. An alignment of human and murine Soggy proteins to human andmurine Dkk-3 proteins is depicted in FIG. 10A-10B.

In one embodiment, a Soggy protein is identified based on the presenceof at least one soggy domain or “SGY” domain in the protein orcorresponding nucleic acid molecule. As defined herein, a “SGY domain”includes a protein domain of a Soggy protein (e.g., hSoggy-1) having anamino acid sequence of about 45-56 amino acids and having at least about25-40% identity with amino acid residues 90-140 of hDkk-3 (leu90-glu140of SEQ ID NO:2). In another embodiment, a “SGY domain” has 46-55,preferably 47-54, more preferably about 48-53, and even more preferablyabout 49-52 or 50-51 amino acids, and has at least about 27-38%,preferably about 28-37%, more preferably about 29-36%, even morepreferably about 30-35%, and even more preferably about 31-34%, or32-33% identity with amino acid residues 90-140 of hDkk-3 (leu90-glu140of SEQ ID NO:2). In yet another embodiment, a “SGY domain” has thefollowing consensus sequence:L-P-X(3)-H-X-E-X(7)-G-N-X-T-X(3)-H-X(4)-K-X-T-X-N-X(2)-G-X(4)-S-E-X-V-X(2)-S-X(4)-E(SEQ ID NO:29). For example, human Soggy-1 has a SGY domain from aboutamino acid residues 81-131 (Leu 81-G1u131 of SEQ ID NO:14) having 33%identity with amino acid residues 90-140 of hDkk-3 (leu90-glu140 of SEQID NO:2). Likewise, murine Soggy-1 has a SGY domain from about aminoacid residues 71-120 (Leu 71-Glu120 of SEQ ID NO:27) having 33% identitywith amino acid residues 90-140 of hDkk-3 (leu90-glu140 of SEQ ID NO:2).The SGY domains of human and murine Soggy-1 are depicted by shaded boxesin FIG. 10A-10B.

In another embodiment of the invention, a Soggy protein has at least oneSGY domain and a signal sequence. For example, in one embodiment, ahSoggy-1 protein contains a signal sequence of about amino acids 1-29,1-30, 1-31, or 1-32 of SEQ ID NO:14. Accordingly, a mature hSoggy-1protein begins at about amino acid residue 30, 31, 32, or 33 of SEQ IDNO:14 and is about 213, 212, 211, or 210 amino acids in length. Inanother embodiment, a mSoggy-1 protein contains a signal sequence ofabout amino acids 1-19, 1-20, 1-21, or 1-22 of SEQ ID NO:27.Accordingly, a mature mSoggy-1 protein begins at about amino acidresidue 211, 210, 209, or 208 of SEQ ID NO:28 and is about 213, 212,211, or 210 amino acids in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode Dkk proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify Dkk-encoding nucleic acids (e.g., Dkk mRVA) andfragments for use as PCR primers for the amplification or mutation ofDkk nucleic acid molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5 and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated Dkk nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived. Anisolated chromosome is not an isolated nucleic acid molecule as definedherein. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention. e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQID NO:7, SEQ ID NO:13, or SEQ ID NO:20. the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98452,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98633, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 207140, ora portion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all orportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7. SEQ ID NO:13, or SEQ ID NO:20, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98452,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98633, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 207140, asa hybridization probe. Dkk nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook. J., Fritsh. E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13. or SEQ ID NO:20, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140, can be isolated by the polymerase chainreaction (PCR) using synthetic oligonucleotide primers designed basedupon the sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:13, or SEQ ID NO:20, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633. or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to Dkk nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1. Thesequence of SEQ ID NO:1 corresponds to the human Dkk-3 cDNA. This cDNAcomprises sequences encoding the human Dkk-3 protein (i.e., “the codingregion”, from nucleotides 38-1087), as well as 5′ untranslated sequences(nucleotides 1 to 37) and 3′ untranslated sequences (nucleotides 1088 to2479). Alternatively, the nucleic acid molecule can comprise only thecoding region of SEQ ID NO:1 (e.g., nucleotides 38 to 1087,corresponding to SEQ ID NO:3). A plasmid containing the full-lengthnucleotide sequence encoding hDkk-3 was deposited with the American TypeCulture Collection (ATCC), presently in Manassas Va., on Jun. 11, 1997and assigned Accession Number 98452.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:4.The sequence of SEQ ID NO:4 corresponds to the human Dkk-4 cDNA. ThiscDNA comprises sequences encoding the human Dkk-4 protein (i.e., “thecoding region”, from nucleotides 125-796), as well as 5′ untranslatedsequences (nucleotides 1 to 124) and 3′ untranslated sequences(nucleotides 797 to 848). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:4 (e.g., nucleotides 125 to796, corresponding to SEQ ID NO:6).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:7.The sequence of SEQ ID NO:7 corresponds to the human Dkk-1 cDNA. ThiscDNA comprises sequences encoding the human Dkk-1 protein (i.e., “thecoding region”, from nucleotides 109-906), as well as 5′ untranslatedsequences (nucleotides 1 to 108) and 3′ untranslated sequences(nucleotides 907-1536). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:7 (e.g., nucleotides109-906, corresponding to SEQ ID NO:9). A plasmid containing thefull-length nucleotide sequence encoding hDkk-1 was deposited with theAmerican Type Culture Collection (ATCC), presently in Manassas Va., onJan. 16, 1998 and assigned Accession Number 98633.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:20.The sequence of SEQ ID NO:20 corresponds to the human Dkk-2 cDNA. ThiscDNA comprises sequences encoding the human Dkk-2 protein (i.e., “thecoding region”, from nucleotides 724-1500), 5′ untranslated sequences(nucleotides 1-723), as well as 3′ untranslated sequences (nucleotides1501-3687). Alternatively, the nucleic acid molecule can comprise onlythe coding region of SEQ ID NO:20 (e.g., nucleotides 724 to 1500,corresponding to SEQ ID NO:22). A plasmid, clone fthu 133, containingthe. full-length nucleotide sequence encoding hDkk-2 was deposited withthe American Type Culture Collection (ATCC), presently in Manassas Va.,on Mar. 2, 1999 and assigned Accession Number 207140.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:13.The sequence of SEQ ID NO:13 corresponds to the human Soggy cDNA. ThiscDNA comprises sequences encoding the human Soggy protein (i.e., “thecoding region”, from nucleotides 75 to 800), as well as 5′ untranslatedsequences (nucleotides 1 to 74) and 3′ untranslated sequences(nucleotides 801 to 928). Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:13 (e.g., nucleotides 75 to800, corresponding to SEQ ID NO:15).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:13, or SEQ ID NO:20, the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, or aportion of any of these nucleotide sequences. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140, is one which is sufficiently complementaryto the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:13, or SEQ ID NO:20, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98452,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number 98633, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 207140,such that it can hybridize to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 30-35%, preferably about 40-45%, more preferably about50-55%, even more preferably about 60-65%, and even more preferably atleast about 70-75%, 80-85%, 90-95% or more homologous to the nucleotidesequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:7. SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:20, orSEQ ID NO:22, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98452, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98633, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 207140, or a portion of any ofthese nucleotide sequences.

In one aspect. the present invention features isolated nucleic acidmolecules which are linear (e.g., linear fragments of double-strandedDNA, linear strands of single-stranded DNA. single-stranded RNAmolecules. and oligonucleotides). Another aspect of the presentinvention features circular nucleic acid molecules (e.g.,double-stranded DNA molecules, for example, plasmid molecules includingthe nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3. SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:20, or SEQ ID NO:22, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, or aportion of any of these nucleotide sequences).

In one embodiment, the isolated nucleic acid molecules of the presentinvention are DNA molecules which are in a form suitable for expression(e.g., suitable for expression of corresponding messenger RNA or mRNA).In another embodiment, the isolated nucleic acid molecules are DNAmolecules which are in a form suitable for expression of correspondingprotein (e.g., in a form, for example, in a vector, which is capable ofexpressing protein, e.g., in the appropriate orientation for expressionfrom regulatory elements and/or in-frame with appropriate regulatoryelements). In another embodiment, the isolated nucleic acids are in aform suitable for determination of nucleic acid sequence (e.g., in aform suitable for sequencing, for example, is a sequencing vectorincluding a M13, T7, T3 and SP6 promoter. Examples of sequencing vectorsinclude, but are not limited to pBluescript (Stratagene™), pT7T3D(Pharmcia™) and pCR2.1 (InVitrogen). In yet another embodiment, theisolated nucelic acid molecules are free from vector sequences. In apreferred embodiment, an isolated nucleic acid molecule is free fromsequencing vector sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of a Dkk protein or Dkk-relatedprotein. The nucleotide sequence determined from the cloning of thehuman Dkk genes allows for the generation of probes and primers designedfor use in identifying and/or cloning Dkk homologues in other celltypes, e.g., from other tissues, as well as Dkk homologues from othermammals and Dkk-related proteins. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, preferably about 25, morepreferably about 40, 50 or 75 consecutive nucleotides of a sensesequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:20, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98452, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98633, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 207140, of an anti-sensesequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ IDNO:20, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98452, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98633, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 207140, or of a naturallyoccurring mutant of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13,SEQ ID NO:20, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98452, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98633, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 207140. In an exemplaryembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which hybridizes under stringent hybridizationconditions to a nucleic acid molecule consisting of nucleotides 470-2479of SEQ ID NO:1 or to a nucleic acid molecule consisting of nucleotides1-475 of SEQ ID NO:4.

Probes based on human nucleotide sequences (e.g.; the human Dkknucleotide sequence) can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. For instance,primers based on the nucleic acid represented in SEQ ID NOs: 1 or 3 canbe used in PCR reactions to clone Dkk homologs (e.g., hDkk-3homologues). In a preferred embodiment of the invention, Dkk homologsare cloned by PCR amplification (e.g., RT-PCR) using primers hybridizingto a portion of the nucleotide sequence encoding the Dkk cysteine richdomain. Likewise, probes based on the subject Dkk sequences can be usedto detect transcripts or genomic sequences encoding the same orhomologous proteins. In preferred embodiments, the probe furthercomprises a label group attached thereto, e.g., the label group can be aradioisotope. a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress a Dkk protein, such as bymeasuring a level of a Dkk-encoding nucleic acid in a sample of cellsfrom a subject e.g., detecting Dkk mRNA levels or determining whether agenomic Dkk gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of a Dkkor Dkk-related protein” can be prepared by isolating a portion of SEQ IDNO:1. SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140, which encodes a polypeptide having abiological activity (the biological activities of the Dkk andDkk-related proteins have previously been described), expressing theencoded portion of the protein (e.g., by recombinant expression invitro) and assessing the activity of the encoded portion of the protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, due todegeneracy of the genetic code and thus encode the same proteins asthose encoded by the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98452, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98633, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 207140. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence shown in SEQ ID NO:2. SEQ ID NO:5. SEQ IDNO:8, SEQ ID NO:14, or SEQ ID NO:21.

In addition to the human nucleotide sequences shown in SEQ ID NO:1, SEQID NO:4, SEQ ID NO:7. SEQ ID NO:13, SEQ ID NO:20. the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98452, the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 98633, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 207140, it will be appreciated by those skilled in theart that DNA sequence polymorphisms that lead to changes in the aminoacid sequences of the Dkk or Dkk-related proteins may exist within apopulation (e.g., the human population). Such genetic polymorphism inthe Dkk or Dkk-related genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a protein, preferably a mammalian Dkk orDkk-related protein. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of a Dkk orDkk-related gene. Any and all such nucleotide variations and resultingamino acid polymorphisms in genes that are the result of natural allelicvariation and that do not alter the functional activity of a Dkk orDkk-related protein are intended to be within the scope of theinvention.

Moreover, nucleic acid molecules encoding Dkk or Dick-related proteinsfrom other species, and thus which have a nucleotide sequence whichdiffers from the human sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, areintended to be within the scope of the invention. For example, a murineDkk-3 cDNA has been identified based of the nucleotide sequence of humanDkk-3. The nucleotide sequence of murine Dkk-3 (SEQ ID NO:16) encodes ahDkk-3 protein having 349 amino acids. The nucleotide and amino acidsequences of murine Dkk-3 are depicted in FIG. 5. The coding region ofmurine Dkk-3 is represented by SEQ ID NO:18. A plasmid containing thefull-length nucleotide sequence encoding mDkk-3 was deposited with theAmerican Type Culture Collection (ATCC), presently in Manassas, Va., onJan. 16, 1998 and assigned Accession Number 98634. Likewise, a murineDkk-related protein (Soggy-1) has been identified based of thenucleotide sequence of human Dkk-3. The nucleotide sequence of murineSoggy-1 (SEQ ID NO:26) encodes a protein having 230 amino acids (SEQ IDNO:27). The nucleotide and amino acid sequences of murine Soggy-1 aredepicted in FIG. 8. The coding region of murine Soggy-1 is representedby SEQ ID NO:28.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the Dkk or Dkk-related cDNAs of the invention can beisolated based on their homology to the human nucleic acids disclosedherein using the human cDNA, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions. Examples of tissues and/or libraries suitablefor isolation of the subject nucleic acids include brain, spinal chordand heart tissue, cDNA encoding a Dkk protein (e.g., a hDkk-3 protein)can be obtained by isolating total mRNA from a cell, e.g., a vertebratecell, a mammalian cell, or a human cell, including embryonic cells.Double stranded cDNAs can then be prepared from the total mMRNA, andsubsequently inserted into a suitable plasmid or bacteriophage vectorusing any one of a number of known techniques. The gene encoding ahDkk-3 protein can also be cloned using established polymerase chainreaction techniques in accordance with the nucleotide sequenceinformation provided by the invention. The nucleic acid of the inventioncan be DNA or RNA or analogs thereof.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 98452, the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98633, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 207140. In anotherembodiment, the nucleic acid is at least 30, 50, 100, 250, 300, 400 or500 nucleotides in length. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 70%,more preferably at least about 80%, even more preferably at least about85% or 90% homologous to each other typically remain hybridized to eachother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. Preferably. an isolated nucleicacid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:1 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

In addition to naturally-occurring allelic variants of the Dkk orDkk-related sequences that may exist in the population, the skilledartisan will further appreciate that changes can be introduced bymutation into the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:4, SEQID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, therebyleading to changes in the amino acid sequence of the encoded Dkkproteins, without altering the functional ability of the Dkk proteins.For example, nucleotide substitutions leading to amino acidsubstitutions (particularly conservative amino acid substitutions) at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20. thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140. A “non-essential” amino acid residue is aresidue that can be altered from the wild-type sequence of Dkk (orwild-type Dkk-related sequence) (e.g., the sequence of SEQ ID NO:2, SEQID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21) without alteringthe biological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare conserved among the Dkk or Dkk-related proteins of the presentinvention (e.g., cysteine residues within cysteine-rich domains), arepredicted to be particularly unamenable to alteration. Furthermore,amino acid residues that are conserved between Dkk protein and otherproteins having cysteine-rich domains are not likely to be amenable toalteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding Dkk or Dkk-related proteins that contain changes inamino acid residues that are not essential for activity. Such proteinsdiffer in amino acid sequence from SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:14, or SEQ ID NO:21 yet retain biological activity. Inone embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a protein, wherein the protein comprises anamino acid sequence at least about 60% homologous to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQID NO:21. Preferably, the protein encoded by the nucleic acid moleculeis at least about 65-70% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:14, or SEQ ID NO:21, more preferably at least about75-80% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:14, or SEQ ID NO:21, even more preferably at least about 85-90%homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, orSEQ ID NO:21, and most preferably at least about 95% homologous to SEQID NO:2. SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21.

An isolated nucleic acid molecule encoding a Dkk protein homologous tothe protein of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, orSEQ ID NO:21 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 207140, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO:1, SEQ ID NO:4. SEQID NO:7. SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98452, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98633, or the nucleotide sequence of the DNA insertof the plasmid deposited with ATCC as Accession Number 207140, bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine. asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, forexample, a predicted nonessential amino acid residue in a Dkk protein(e.g., one not located in a cysteine-rich domain) is preferably replacedwith another amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a Dkk or Dkk-related coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 98452, the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98633, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 207140, the encodedprotein can be expressed recombinantly and the activity of the proteincan be determined.

In a preferred embodiment, a mutant Dkk or Dkk-related protein can beassayed for intracellular calcium, an increase in phosphatidylinositolor other molecule, and can result, e.g., in phosphorylation of specificproteins, a modulation of gene transcription and any of the otherbiological activities set forth herein.

In a preferred embodiment, a mutant Dkk or Dkk-related protein can alsobe assayed for the ability to (1) modulate cellular signal transduction,either in vitro or in vivo; (2) regulate communication between cells;(3) regulate expression of genes whose expression is modulated bybinding of Dkk (e.g., hDkk-3) to a receptor; (4) regulate genetranscription in a cell involved in development or differentiation,either in vitro or in vivo; (5) regulate cellular proliferation, eitherin vitro or in vivo; (6) form and/or maintain ordered spatialarrangements of differentiated tissues in vertebrates; (7) modulate celldeath (e.g. cell survival); (8) regulate cell migration; and/or (9)modulate immune system function.

In addition to the nucleic acid molecules encoding Dkk or Dkk-relatedproteins described above, another aspect of the invention pertains toisolated nucleic acid molecules which are antisense thereto. An“antisense” nucleic acid comprises a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire Dkk coding strand, or to only aportion thereof. In one embodiment, an antisense nucleic acid moleculeis antisense to a “coding region” of the coding strand of a nucleotidesequence encoding Dkk. The term “coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated intoamino acid residues (e.g., the coding region of human Dkk-3 correspondsto SEQ ID NO:3, the coding region of human Dkk-4 corresponds to SEQ IDNO:6, the coding region of human Dkk-1 corresponds to SEQ ID NO:9, thecoding region of human Dkk-2 corresponds to SEQ ID NO:22. and the codingregion of human Soggy corresponds to SEQ ID NO:15). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding a Dkk or Dkk-related protein. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Given the coding strand sequences disclosed herein (e.g., SEQ ID NO:3.SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:22). antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of a Dkk or Dkk-relatedmRNA, but more preferably is an oligonucleotide which is antisense toonly a portion of the coding or noncoding region of the mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of Dkk mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 nucleotides in length. Ina preferred embodiment, an oligonucleotide is about 30-90, preferablyabout 40-80, more preferably about 50-70 nucleotides in length and isantisense to a portion of SEQ ID NO:1 from about nucleotides 1-150. Inanother embodiment, an oligonucleotide is antisense to a portion of SEQID NO:4 from about nucleotides 25-225. In another embodiment, anoligonucleotide is antisense to a portion of SEQ ID NO:7 from aboutnucleotides 1-200. In another embodiment, an oligonucleotide isantisense to a portion of SEQ ID NO:20 from about nucleotides 625-825.In yet another embodiment, an oligonucleotide is antisense to a portionof SEQ ID NO:13 from about nucleotides 1-175.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives, acridine substitutednucleotides, can be used. Alternatively, the antisense nucleic acidmolecule can by synthesized to increase transport across cellularmembranes, e.g., methylphosphonate derivatives. The antisense moleculescan include a 3′-terminal cap (e.g., a 3′-aminopropyl modification), abiotin moiety. or even a 3′-3′ terminal linkage.

Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethyl guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil,5-methoxyuracil-methylthio-N-6-isopentenyladenine, uracil-5-oxyaceticacid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2.6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a Dkk orDkk-related protein to thereby inhibit expression of the protein, e.g.,by inhibiting transcription and/or translation. The hybridization can beby conventional nucleotide complementarity to form a stable duplex, or,for example, in the case of an antisense nucleic acid molecule whichbinds to DNA duplexes, through specific interactions in the major grooveof the double helix. An example of a route of administration ofantisense nucleic acid molecules of the invention include directinjection at a tissue site. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBSLett. 215:327-330).

In still another embodiment. an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveDkk or Dkk-related mRNA transcripts to thereby inhibit translation ofDkk or Dkk-related mRNA. A ribozyme having specificity for a Dkk- orDkk-related-encoding nucleic acid can be designed based upon thenucleotide sequence of a Dkk or Dkk-related cDNA disclosed herein (i.e.,SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98452, the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98633). For example,a derivative of a Tetrahymena L-19 IVS RNA can be constructed in whichthe nucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a Dkk-encoding mRNA. See, e.g.,Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat. No.5,116,742. Alternatively, Dkk (or Dkk-related) mRNA can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418.

Alternatively, gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the Dkk orDkk-related gene (e.g., the promoter and/or enhancers) to form triplehelical structures that prevent transcription of the gene in targetcells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al., (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the nucleic acid molecules of the presentinvention can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acid molecules can be modified to generate peptide nucleic acids(see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4(1):5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” referto nucleic acid mimics. e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAs hasbeen shown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. PNAS93:14670-675.

PNAs of Dkk or Dkk-related nucleic acid molecules can be usedtherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of Dkk or Dkk-related nucleic acidmolecules can also be used in the analysis of single base pair mutationsin a gene, (e.g., by PNA-directed PCR clamping); as ‘artificialrestriction enzymes’ when used in combination with other enzymes, (e.g.,S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

In another embodiment. PNAs of Dkk can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of Dkk nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810, published Dec. 15, 1988) or the blood-brainbarrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25,1988). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (See, e.g., Krol et al. (1988)BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988)Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, (e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

II. Isolated Dkk Proteins and Anti-Dkk Antibodies

One aspect of the invention pertains to isolated Dkk proteins,Dkk-related proteins and biologically active portions thereof, as wellas polypeptide fragments suitable for use as immunogens to raiseantibodies. In one embodiment, native Dkk or Dkk-related proteins can beisolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a Dkk or Dkk-related protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which the Dkkor Dkk-related protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of Dkk or Dkk-related protein having less thanabout 30% (by dry weight) of non-Dkk protein or non-Dkk-related protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-Dkk protein or non-Dkk-related protein, stillmore preferably less than about 10% of non-Dkk protein ornon-Dkk-related protein, and most preferably less than about 5% non-Dkkprotein or non-Dkk-related protein. When the Dkk or Dkk-related proteinor biologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of Dkk or Dkk-related protein in whichthe protein is separated. from chemical precursors or other chemicalswhich are involved in the synthesis of the protein. In one embodiment,the language “substantially free of chemical precursors or otherchemicals” includes preparations of Dkk protein having less than about30% (by dry weight) of chemical precursors, non-Dkk chemicals, ornon-Dkk-related chemicals, more preferably less than about 20% chemicalprecursors, non-Dkk chemicals, or non-Dick-related chemicals, still morepreferably less than about 10% chemical precursors, non-Dkk chemicals,or non-Dkk-related chemicals, and most preferably less than about 50%chemical precursors, non-Dick chemicals, or non-Dkk-related chemicals.

Biologically active portions of a Dkk or Dkk-related protein includepeptides comprising amino acid sequences sufficiently homologous to orderived from the amino acid sequence of the Dkk or Dick-related protein,e.g., the amino acid sequence shown in SEQ ID NO:2, SEQ ID.NO:5, SEQ IDNO:8. SEQ ID NO:14, or SEQ ID NO:21, which include less amino acids thanthe full length proteins, and exhibit at least one activity of a Dkk orDkk-related protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the Dick or Dkk-relatedprotein. A biologically active portion of a protein can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.

In one embodiment. a biologically active portion of a Dkk proteincomprises at least a cysteine-rich region. In another embodiment, abiologically active portion of a Dkk protein comprises at least acysteine-rich region, wherein the cysteine-rich region includes at leastone cysteine-rich domain. In yet another embodiment, a biologicallyactive portion of a Dkk protein comprises at least a signal sequence.

In another embodiment, a biologically active portion of a Dkk-relatedprotein (e.g., a Soggy protein) comprises at least a Soggy domain. Inyet another embodiment, a biologically active portion of a Dkk-relatedprotein comprises at least a signal sequence.

In an alternative embodiment, a biologically active portion of a Dkk orDkk-related protein comprises an amino acid sequence lacking a signalsequence.

It is to be understood that a preferred biologically active portion of aDkk or Dkk-related protein of the present invention may contain at leastone of the above-identified structural domains. A more preferredbiologically active portion of a Dkk or Dkk-related protein may containat least two of the above-identified structural domains. An even morepreferred biologically active portion of a protein may contain at leastthree of the above-identified structural domains. A particularlypreferred biologically active portion of a protein of the presentinvention may contain at least four of the above-identified structuraldomains.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native Dkkor Dkk-related protein.

In a preferred embodiment, the Dkk protein has an amino acid sequenceshown in SEQ ID NO:2 or an amino acid sequence at least about 55%homologous to SEQ ID NO:2. In another preferred embodiment, the Dkkprotein has an amino acid sequence shown in SEQ ID NO:5 or an amino acidsequence at least about 35% homologous to SEQ ID NO:5. In anotherpreferred embodiment, the Dkk protein has an amino acid sequence shownin SEQ ID NO:8 or an amino acid sequence at least about 85% homologousto SEQ ID NO:8. In another preferred embodiment, the Dkk protein has anamino acid sequence shown in SEQ ID NO:21 or an amino acid sequence atleast about 35% homologous to SEQ ID NO:21. In another preferredembodiment, the protein has an amino acid sequence shown in SEQ ID NO:14or an amino acid sequence at least about 60% homologous to SEQ ID NO:14.In still another preferred embodiment, a protein of the presentinvention comprises an amino acid sequence which is at least about30-35%, preferably about 40-45%, more preferably about 50-55%, even morepreferably about 60-65%, and even more preferably at least about 70-75%,80-85%, 90-95% or more homologous to the amino acid sequences shown inSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21.

In other embodiments, the protein is substantially homologous to SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, and,preferably. retains the functional activity of the protein of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail in subsection I above. Accordingly,in another embodiment, the protein is a protein which comprises an aminoacid sequence at least about 60% homologous to the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21and. preferably, retains the functional activity of the proteins of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:11,respectively. Preferably, the protein is at least about 70% homologousto SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21,more preferably at least about 80% homologous to SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, even more preferablyat least about 90% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,SEQ ID NO:14, or SEQ ID NO:21, and most preferably at least about 95% ormore homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14,or SEQ ID NO:21.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence and non-homologous sequences can be disregardedfor comparison purposes). In one embodiment, an alignment is a globalalignment, e.g., an overall sequence alignment. In another embodiment,an alignment is a local alignment. In a preferred embodiment, the lengthof a sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, or90% of the length of the reference sequence to which it is aligned(e.g., when aligning a second sequence to the Dkk amino acid sequence ofSEQ ID NO:2, at least 105, preferably at least 145, more preferably atleast 175, even more preferably at least 210, and even more preferablyat least 245, 280 or 315 amino acid residues are aligned). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each cap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at www.gcg.com), usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, 5, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available at www.gcg.com), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1. 2, 3, 4,5, or 6. In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM 120weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to Dkk nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program. score=50,wordlength=3 to obtain amino acid sequences homologous to Dkk proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlrn.nih.gov.

The invention also provides Dkk or Dkk-related chimeric or fusionproteins. As used herein, a “chimeric protein” or “fusion protein”comprises a Dkk or Dkk-related polypeptide operatively linked to anon-Dkk polypeptide or non-Dkk-related polypeptide. A “Dkk polypeptide”or “Dkk-related polypeptide” refers to a polypeptide having an aminoacid sequence corresponding to Dkk or a Dkk-related protein, whereas a“non-Dkk polypeptide” or “non-Dkk-related polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homoloeous to the Dkk or Dkk-related protein,e.g., a protein which is different from the Dkk or Dkk-related proteinand which is derived from the same or a different organism. Within a Dkkor Dkk-related fusion protein the Dkk or Dkk-related polypeptide cancorrespond to all or a portion of a Dkk or Dick-related protein. In apreferred embodiment, a Dick or Dkk-related fusion protein comprises atleast one biologically active portion of a Dkk protein. In anotherpreferred embodiment, a Dkk or Dkk-related fusion protein comprises atleast two biologically active portions of a Dkk or Dkk-related protein.In another preferred embodiment, a Dick or Dkk-related fusion proteincomprises at least three biologically active portions of a Dkk orDkk-related protein. Within the fusion protein, the term “operativelylinked” is intended to indicate that the Dick or Dick-relatedpolypeptide and the non-Dick or non-Dick-related polypeptide are fusedin-frame to each other. The non-Dkk or non-Dick-related polypeptide canbe fused to the N-terminus or C-terminus of the Dkk or Dkk-relatedpolypeptide.

For example, in one embodiment, the fusion protein is a GST-Dkk fusionprotein in which the Dick sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant Dkk.

In another embodiment, the fusion protein is a Dkk or Dkk-relatedprotein containing a heterologous signal sequence at its N-terminus. Forexample, the native Dick signal sequence (i.e., about amino acids 1 to23 of SEQ ID NO:2) can be removed and replaced with a signal sequencefrom another protein. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of Dkk or Dkk-related proteins canbe increased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a Dkk-immunoglobulinfusion protein in which the Dkk sequences comprising primarily the Dkkcysteine-rich regions are fused to sequences derived from a member ofthe immunoglobulin protein family. Soluble derivatives have also beenmade of cell surface glycoproteins in the immunoglobulin genesuperfamily consisting of an extracellular domain of the cell surfaceglycoprotein fused to an immunoglobulin constant (Fc) region (see e.g.,Capon, et al. (1989) Nature 337:525-531 and Capon U.S. Pat. Nos.5,116,964 and 5,428,130 [CD4-IgG1 constructs]; Linsley, P. S. et al.(1991) J. Exp. Med. 173:721-730 [a CD28-IgG1 construct and a B7-1-IgG1construct]; and Linsley, P. S. et al. (1991) J. Exp. Med. 174:561-569and U.S. Pat. No. 5,434,131 [a CTLA4-IgG1]). Such fusion proteins haveproven useful for modulating receptor-ligand interactions. Solublederivatives of cell surface proteins of the tumor necrosis factorreceptor (TNFR) superfamily proteins have been made consisting of anextracellular domain of the cell surface receptor fused to animmunoglobulin constant (Fc) region (See for example Moreland et al.(1997) N. Engl. J. Med. 337(3):141-147; van der Poll et al. (1997) Blood89(10):3727-3734; and Ammann et al. (1997) J. Clin. Invest.99(7):1699-1703.)

The Dkk-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a Dkk ligand and a Dkkreceptor on the surface of a cell, to thereby suppress Dkk-mediatedsignal transduction in vivo. The Dkk-immunoglobulin fusion proteins canbe used to affect the bioavailability of a Dkk cognate receptor.Inhibition of the Dkk ligand Dkk interaction may be usefultherapeutically for both the treatment of differentiative orproliferative disorders, as well as modulating (e.g., promoting orinhibiting) developmental responses, cell adhesion, and/or cell fate.Moreover, the Dkk-immunoglobulin fusion proteins of the invention can beused as immunogens to produce anti-Dkk antibodies in a subject, topurify Dkk ligands and in screening assays to identify molecules whichinhibit the interaction of Dkk with a Dkk ligand.

Preferably. a Dkk or Dkk-related chimeric or fusion protein of theinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel etal., John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A Dkk-encoding nucleic acid or nucleic acid encoding aDkk-related protein can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the protein.

The present invention also pertains to variants of the Dkk orDkk-related proteins which function as either agonists (mimetics) or asantagonists. Variants of the Dkk or Dkk-related proteins can begenerated by mutagenesis, e.g., discrete point mutation or truncation ofa Dkk or Dkk-related protein. An agonist of the Dkk or Dkk-relatedproteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a Dkk orDkk-related protein. An antagonist of a Dkk or Dkk-related protein caninhibit one or more of the activities of the naturally occurring form ofthe protein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade which includes the Dkkor Dkk-related protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. In oneembodiment, treatment of a subject with a variant having a subset of thebiological activities of the naturally occurring form of the protein hasfewer side effects in a subject relative to treatment with the naturallyoccurring form of the Dkk or Dkk-related protein.

In one embodiment, variants of a Dkk or Dkk-related protein whichfunction as either agonists (mimetics) or as antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of a Dkk or Dkk-related protein for protein agonistor antagonist activity. In one embodiment, a variegated library of Dkkor Dkk-related variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of Dkk or Dkk-related variants can be produced by,for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential Dkk or Dkk-related sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of Dkk or Dkk-relatedsequences therein. There are a variety of methods which can be used toproduce libraries of potential Dkk or Dkk-related variants from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential Dkk orDkk-related sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang. S. A. (1983)Tetrahedron 39:3: Itakura et al., (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056: Ike et al., (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of a Dkk or Dkk-related proteincoding sequence can be used to generate a variegated population of Dickor Dkk-related fragments for screening and subsequent selection ofvariants of a Dkk or Dkk-related protein. In one embodiment, a libraryof coding sequence fragments can be generated by treating a doublestranded PCR fragment of a Dkk coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the Dkk protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of Dkk or Dkk-relatedproteins. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recrusive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify Dkk variants (Arkin and Yourvan (1992) PNAS89:7811-7815; Delgrave et al., (1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated Dkk or Dkk-related library. For example, a library ofexpression vectors can be transfected into a cell line which ordinarilyresponds to a particular ligand in a Dkk-dependent manner. Thetransfected cells are then contacted with the ligand and the effect ofexpression of the mutant on signaling by the ligand can be detected,e.g., by measuring any of a number of immune cell responses. Plasmid DNAcan then be recovered from the cells which score for inhibition, oralternatively, potentiation of ligand induction, and the individualclones further characterized.

An isolated Dkk protein, Dkk-related protein, or a portion or fragmentthereof, can be used as an immunogen to generate antibodies that bindDkk or Dkk-related proteins using standard techniques for polyclonal andmonoclonal antibody preparation. A full-length Dkk or Dkk-relatedprotein can be used or, alternatively, the invention provides antigenicpeptide fragments for use as immunogens. The antigenic peptide of Dkkcomprises at least 8 amino acid residues of the amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ IDNO:21 and encompasses an epitope of Dkk or Dkk-related protein such thatan antibody raised against the peptide forms a specific immune complexwith the protein. Preferably, the antigenic peptide comprises at least10 amino acid residues, more preferably at least 15 amino acid residues,even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofDkk or Dkk-related proteins that are located on the surface of theprotein, e.g., hydrophilic regions.

A Dkk or Dkk-related immunogen typically is used to prepare antibodiesby immunizing a suitable subject, (e.g., rabbit, coat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed Dkk or Dkk-related proteinor a chemically synthesized Dkk or Dkk-related polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. Immunizationof a suitable subject with an immunogenic Dkk preparation, for example,induces a polycional anti-Dkk antibody response.

Accordingly, another aspect of the invention pertains to anti-Dkkantibodies as well as antobodies to Dkk-related proteins. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, such as Dkk or Dkk-related antigens.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind Dkk or Dkk-relatedpolypeptides. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of Dkk or a orDkk-related protein. A monoclonal antibody composition thus typicallydisplays a single binding affinity for a particular Dkk or Dkk-relatedprotein with which it immunoreacts.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a Dkk or Dkk-related immunogen. The antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized Dkk or Dkk-related protein. If desired. the antibodymolecules directed against Dkk or Dkk-related protein can be isolatedfrom the mammal (e.g., from the blood) and further purified by wellknown techniques, such as protein A chromatography to obtain the IgGfraction. At an appropriate time after immunization, e.g., when theantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-447) (see also, Brown et al.,(1981) J. Immunol. 127:539-46; Brown et al., (1980) J. Biol. Chem.255:4980-83; Yeh et al., (1976) PNAS 76:2927-31; and Yeh et al., (1982)Int. J. Cancer 29:269-75}, the more recent human B cell hybridomatechnique (Kozbor et al., (1983) Immunol Today 4:72), the EBV-hybridomatechnique (Cole et al., (1985), Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.,(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a Dkk or Dkk-related immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds Dkk or Dkk-related protein.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody (see, e.g., G. Galfre et al., (1977) Nature266:55052; Gefter et al., Somatic Cell Genet. cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindDkk or Dkk-related protein, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with Dkk or Dkk-related protein to therebyisolate immunoglobulin library members that bind Dkk or Dkk-relatedprotein. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example. Ladner et al.,U.S. Pat. No. 5,223,409; Kang et al., PCT International Publication No.WO 92/18619; Dower et al., PCT International Publication No. WO91/17271; Winter et al., PCT International Publication WO 92/20791;Markland et al., PCT International Publication No. WO 92/15679;Breitling et al., PCT International Publication WO 93/01288; McCaffertyet al., PCT International Publication No. WO 92/01047; Garrard et al.,PCT International Publication No. WO 92/09690; Ladner et al., PCTInternational Publication No. WO 90/02809; Fuchs et al., (1991)Bio/Technology 9:1370-1372; Hay et al., (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffiths et al.,(1993) EMBO J. 12:725-734; Hawkins et al., (1992) J. Mol. Biol.226:889-896; Clarkson et al., (1991) Nature 352:624-628; Gram et al.,(1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio/Technology9:1373-1377; Hoogenboom et al., (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al., (1991) PNAS 88:7978-7982; and McCafferty et al., Nature(1990) 348:552-554.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in Robinson et al.,International Application No. PCT/US86/02269; Akira, et al., EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., PCT International Publication No. WO 86/01533: Cabilly et al.,U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application125,033; Better et al., (1988) Science 240:1041-1043; Liu et al., (1987)PNAS 84:3439-3443; Liu et al., (1987) J. Immunol. 139:3521-3526; Sun etal., (1987) PNAS 84:214-218; Nishimura et al., (1987) Canc. Res.47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al.,(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al., (1986) BioTechniques 4:214; WinterU.S. Pat. No. 5,225,539; Jones et al., (1986) Nature 321:552-525;Verhoeyan et al., (1988) Science 239:1534; and Beidler et al., (1988) J.Immunol. 141:4053-4060.

An antibody (e.g., monoclonal antibody) can be used to isolate Dkk orDkk-related protein by standard techniques, such as affinitychromatography or immunoprecipitation. An antibody can facilitate thepurification of natural Dkk or Dkk-related protein from cells and ofrecombinantly produced Dkk or Dkk-related protein expressed in hostcells. Moreover, an antibody can be used to detect Dkk or Dkk-relatedprotein (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the Dkk orDkk-related protein. Antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidinfbiotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding Dkk or a nucleicacid encoding a Dkk-related protein (or a portion thereof). As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification. “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses. adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., Dkk proteins, Dkk-relatedproteins, mutant forms of Dkk or Dkk-related proteins, fusion proteins,etc.).

The recombinant expression vectors of the invention can be designed forexpression of Dkk or Dkk-related proteins in prokaryotic or eukaryoticcells. For example, Dkk can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel.Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40). pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in activity assays, in ligandbinding (e.g., direct assays or competitive assays described in detailbelow), to generate antibodies specific for Dkk or Dkk-related proteins,as examples. In a preferred embodiment, a Dkk or Dkk-related fusionexpressed in a retroviral expression vector of the present invention canbe utilized to infect bone marrow cells which are subsequentlytransplanted into irradiated recipients. The pathology of the subjectrecipient is then examined after sufficient time has passed (e.g. six(6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology Methods in Enzymology 185, AcademicPress, San Diego. Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gnl). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gnl gene under the transcriptional control ofthe lacUVS promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman. S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego. Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids.Res. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment. the Dkk or Dkk-related expression vector is ayeast expression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec 1 (Baldari, et al., (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego. CA).

Alternatively, Dkk or Dkk-related protein can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., (1983) Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al., (1987) EMBO J. 6:187-195). Whenused in mammalian cells, the expression vector's control functions areoften provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukarvotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to Dkk mRNA or a Dkk-related mRNA. Regulatorysequences operatively linked to a nucleic acid cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types. for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region. the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, Dkkprotein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation. DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al., (MolecularCloning: A Laboratory Manual, 2nd. ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells. it is known that. dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding Dkk or can be introduced on a separate vector.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a Dkk orDkk-related protein. Accordingly, the invention further provides methodsfor producing Dkk or Dkk-related proteins using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of invention (into which a recombinant expression vector encodingDkk or a Dkk-related protein has been introduced) in a suitable mediumsuch that protein is produced. In another embodiment, the method furthercomprises isolating Dkk or a Dkk-related protein from the medium or thehost cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichDkk-coding sequences (or Dkk-related coding sequences) have beenintroduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous Dkk sequences (or Dkk-relatedsequences) have been introduced into their genome or homologousrecombinant animals in which endogenous Dkk sequences (or Dkk-relatedsequences) have been altered. Such animals are useful for studying thefunction and/or activity of Dkk or Dkk-related proteins and foridentifying and/or evaluating modulators of Dkk or Dkk-related proteinactivity. As used herein, a “transgenic animal” is a non-human animal,preferably a mammal, more preferably a rodent such as a rat or mouse, inwhich one or more of the cells of the animal includes a transgene. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens, amphibians. etc. A transgene is exogenous DNAwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal,thereby directing the expression of an encoded gene product in one ormore cell types or tissues of the transgenic animal. As used herein, a“homolocous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse. in which an endogenous Dkk orDkk-related gene has been altered by homologous recombination betweenthe endogenous gene and an exogenous DNA molecule introduced into a cellof the animal, e.g., an embryonic cell of the animal, prior todevelopment of the animal.

A transgenic animal of the invention can be created, for example. byintroducing Dkk-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The human Dkk cDNA sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, orSEQ ID NO:20 can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homologue of a human Dkkgene, such as a mouse Dkk gene, can be isolated based on hybridizationto the human Dkk cDNA (described further in subsection I above) and usedas a transgene. Intronic sequences and polyadenylation signals can alsobe included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the Dkk transgene to direct expression of Dkk protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al., and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the Dkk transgene in its genome and/or expression of DkkmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding Dkk canfurther be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a Dkk gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the Dkk gene. The Dkk gene can be a human gene(e.g., the cDNA of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ IDNO:22), but more preferably, is a non-human homologue of a human Dkkgene. For example, a mouse Dkk gene of SEQ ID NO:16 can be used toconstruct a homologous recombination vector suitable for altering anendogenous Dkk gene in the mouse genome. In a preferred embodiment. thevector is designed such that, upon homologous recombination, theendogenous Dkk gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).Alternatively, the vector can be designed such that, upon homologousrecombination. the endogenous Dkk gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenousDkk protein). In the homologous recombination vector, the alteredportion of the Dkk gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the Dkk gene to allow for homologous recombination tooccur between the exogenous Dkk gene carried by the vector and anendogenous Dkk gene in an embryonic stem cell. The additional flankingDkk nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3 ends) are included in the vector (seee.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced Dkk gene has homologously recombinedwith the endogenous Dkk gene are selected (see e.g., Li, E. et al.,(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152), A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al. It is also within the scope of the presentinvention to practice the above-described transgenic methodologyutilizing nucleic acid molecules which encode Dkk-related proteins.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system. see, e.g., Lakso et al., (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al., (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut. I. et al., (1997)Nature 385:810-813. In brief, a cell. e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

IV. Pharmaceutical Compositions

The Dkk and Dkk-related nucleic acid molecules, Dkk and Dkk-relatedproteins, and anti-Dkk or anti-Dkk-related protein antibodies (alsoreferred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral. e.g., intravenous, intradermal,subcutaneous. oral (e.g., inhalation). transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents: antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose, pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water. CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,ehlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a Dkk protein, Dkk-related protein or antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack. ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The molecules of the present invention (e.g., nucleic acid molecules,proteins, protein homologues, and antibodies described herein) can beused in one or more of the following methods: a) screening assays; b)predictive medicine (e.g., diagnostic assays, prognostic assays,monitoring clinical trials, and pharmacogenetics); and c) methods oftreatment (e.g., therapeutic and prophylactic). As described herein, aDkk protein of the invention has one or more of the followingactivities: intracellular calcium. an increase in phosphatidylinositolor other molecule, and can result, e.g., in phosphorylation of specificproteins, a modulation of gene transcription and any of the otherbiological activities set forth herein.

In a preferred embodiment, a Dkk activity is at least one or more of thefollowing activities: (i) interaction of a Dkk protein with and/orbinding to a second molecule, (e.g., a protein. such as a Dkk (e.g.,hDkk-3) receptor, a soluble form of a Dkk receptor, a receptor for amember of the wnt family of signaling proteins, or a non-Dkk signalingmolecule); (ii) interaction of a Dkk protein with an intracellularprotein via a membrane-bound Dkk receptor; (iii) complex formationbetween a soluble Dkk protein and a second soluble Dkk binding partner(e.g., a non-Dkk protein molecule or a second Dkk protein molecule);(iv) interaction with other extracellular proteins (e.g., regulation ofwnt-dependent cellular adhesion to extracellular matrix components); (v)binding to and eliminating an undesirable molecule (e.g., a detoxifyingactivity or defense function); and/or (vi) an enzymatic activity, andcan thus be used in, for example, (1) modulation of cellular signaltransduction, either in vitro or in vivo (e.g., antagonism of theactivity of members of the wnt family of secreted proteins orsuppression of wnt-dependent signal transduction); (2) regulation ofcommunication between cells (e.g., regulation of wnt-dependent cell-cellinteractions); (3) regulation of expression of genes whose expression ismodulated by binding of Dkk (e.g., hDkk-3) to a receptor; (4) regulationof gene transcription in a cell involved in development ordifferentiation, either in vitro or in vivo (e.g., induction of cellulardifferentiation); (5) regulation of gene transcription in a cellinvolved in development or differentiation, wherein at least one geneencodes a differentiation-specific protein; (6) regulation of genetranscription in a cell involved in development or differentiation,wherein at least one gene encodes a second secreted protein; (7)regulation of gene transcription in a cell involved in development ordifferentiation, wherein at least one gene encodes a signal transductionmolecule; (8) regulation of cellular proliferation, either in vitro orin vivo (e.g., induction of cellular proliferation or inhibition ofproliferation, for example, inhibition of tumorigenesis (e.g.,inhibition of glioblastoma proliferation)); (9) formation andmaintenance of ordered spatial arrangements of differentiated tissues invertebrates, both adult and embryonic (e.g., induction of head formationduring vertebrate development or maintenance of hematopoietic progenitorcells); (10) modulation of cell death, such as stimulation of cellsurvival; (11) regulating cell migration; and or (12) immune modulation.

Accordingly one embodiment of the present invention involves a method ofuse (e.g., a diagnostic assay, prognostic assay, or aprophylactic/therapeutic method of treatment) wherein a molecule of thepresent invention (e.g., a Dkk protein, Dkk nucleic acid, or a Dkkmodulator) is used, for example, to diagnose, prognose and/or treat adisease and/or condition in which any of the aforementioned activities(i.e., activities (i)-(vi) and (1)-(12) in the above paragraph) isindicated, in another embodiment, the present invention involves amethod of use (e.g., a diagnostic assay, prognostic assay, or aprophylactic/therapeutic method of treatment) wherein a molecule of thepresent invention (e.g., a Dkk protein, Dkk nucleic acid, or a Dkkmodulator) is used, for example, for the diagnosis, prognosis, and/ortreatment of subjects, preferably a human subject, in which any of theaforementioned activities is pathologically perturbed. In a preferredembodiment, the methods of use (e.g., diagnostic assays, prognosticassays, or prophylactic/therapeutic methods of treatment) involveadministering to a subject, preferably a human subject, a molecule ofthe present invention (e.g., a Dkk protein, Dkk nucleic acid, or a Dkkmodulator) for the diagnosis, prognosis, and/or therapeutic treatment.In another embodiment, the methods of use (e.g., diagnostic assays,prognostic assays, or prophylactic/therapeutic methods of treatment)involve administering to a human subject a molecule of the presentinvention (e.g., a Dkk protein, Dkk nucleic acid, or a Dkk modulator).

Other embodiments of the invention pertain to the use of isolatednucleic acid molecules of the invention can be used, for example, toexpress Dkk or Dkk-related protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect Dkk orDkk-related mRNA (e.g., in a biological sample) or a genetic alterationin a Dkk or Dick-related gene, and to modulate Dkk or Dick-relatedactivity, as described further below. In addition, the Dkk orDkk-related proteins can be used to screen drugs or compounds whichmodulate the Dkk activity as well as to treat disorders characterized byinsufficient or excessive production of Dkk or Dkk-related protein orproduction of Dkk or Dkk-related protein forms which have decreased oraberrant activity compared to Dkk or Dkk-related wild type protein(e.g., developmental disorders or proliferative diseases such as canceras well as diseases, ocular disorders (e.g., blindness) conditions ordisorders characterized by abnormal cell differentiation and/orsurvival, an abnormal extracellular structure, or an abnormality in adefense mechanism). Moreover, the antibodies of the invention can beused to detect and isolate Dkk or Dkk-related proteins, regulate thebioavailability of Dkk or Dkk-related proteins, and modulate Dkk orDkk-related activity. The term “an aberrant activity”, as applied to anactivity of a protein such as Dkk (e.g., hDkk-3), refers to an activitywhich differs from the activity of the wild-type or native protein orwhich differs from the activity of the protein in a healthy subject. Anactivity of a protein can be aberrant because it is stronger than theactivity of its native counterpart. Alternatively, an activity can beaberrant because it is weaker or absent related to the activity of itsnative counterpart. An aberrant activity can also be a change in anactivity. For example an aberrant protein can interact with a differentprotein relative to its native counterpart. A cell can have an aberrantDkk (e.g., hDkk-3) activity due to overexpression or underexpression ofthe gene encoding Dkk.

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to Dkk or Dkk-related proteins or have a stimulatory orinhibitory effect on, for example, Dkk or Dkk-related expression oractivity. Modulators can include, for example, agonists and/orantagonists. The term “agonist”, as used herein, is meant to refer to anagent that mimics or upregulates (e.g. potentiates or supplements) a Dkkor Dkk-related (e.g., hDkk-3) bioactivity. An agonist can be a compoundwhich mimics a bioactivity of a Dkk or Dkk-related protein, such astransduction of a signal from a Dkk receptor, by, e.g., interacting witha hDkk-3 receptor. An agonist can also be a compound that upregulatesexpression of a Dkk or Dkk-related gene. An agonist can also be acompound which modulates the expression or activity of a protein whichis located downstream, for example, of a Dkk receptor, thereby mimickingor enhancing the effect of binding of Dkk to a Dkk receptor.

“Antagonist” as used herein is meant to refer to an agent that inhibits,decreases or suppresses a bioactivity (e.g., hDkk-3). An antagonist canbe a compound which decreases signalling from a Dkk or Dkk-relatedprotein, e.g., a compound that is capable of binding to hDkk-3 or to ahDkk-3 receptor. A preferred antagonist inhibits the interaction betweena Dick or Dkk-related protein and another molecule, such as a Dkkreceptor. Alternatively, an antagonist can be a compound thatdownregulates expression of a Dkk or Dkk-related gene. An antagonist canalso be a compound which modulates the expression or activity of aprotein which is located downstream of a Dkk receptor, therebyantagonizing the effect of binding of Dkk to a Dkk receptor.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a Dkk orDkk-related protein or polypeptide or biologically active portionthereof. In another embodiment, the invention provides assays forscreening candidate or test compounds which bind to or modulate theactivity of a Dkk receptor. The test compounds of the present inventioncan be obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.,(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409). plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a Dkk receptor on the cell surface is contacted with a testcompound and the ability of the test compound to bind to a Dkk receptordetermined. The cell, for example, can be of mammalian origin or a yeastcell. Determining the ability of the test compound to bind to a Dkkreceptor can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the Dkk receptor can be determined by detecting the labeledcompound in a complex. For example, test compounds can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a test compound to interact with a Dkk receptor without the labelingof any of the interactants. For example, a microphysiometer can be usedto detect the interaction of a test compound with a Dkk receptor withoutthe labeling of either the test compound or the receptor. McConnell, H.M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor™) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween ligand and receptor.

In a preferred embodiment, the assay comprises contacting a cell whichexpresses a Dkk receptor on the cell surface with a Dkk protein orbiologically-active portion thereof, to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a Dkk receptor, whereindetermining the ability of the test compound to interact with a Dkkreceptor comprises determining the ability of the test compound topreferentially bind to the Dkk receptor as compared to the ability ofDkk, or a biologically active portion thereof, to bind to the receptor.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a Dkk target molecule with a test compoundand determining the ability of the test compound to modulate (e.g.stimulate or inhibit) the activity of the Dkk target molecule.Determining the ability of the test compound to modulate the activity ofa Dkk target molecule can be accomplished, for example, by determiningthe ability of the Dkk protein to bind to or interact with the Dkktarget molecule.

Determining the ability of the Dkk protein to bind to or interact with aDkk target molecule can be accomplished by one of the methods describedabove for determining direct binding. In a preferred embodiment,determining the ability of the Dkk protein to bind to or interact with aDkk target molecule can be accomplished by determining the activity ofthe target molecule. For example, the activity of the target moleculecan be determined by detecting induction of a cellular second messengerof the target (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.),detecting catalytic/enzymatic activity of the target an appropriatesubstrate, detecting the induction of a reporter gene (comprising aDkk-responsive regulatory element operatively linked to a nucleic acidencoding a detectable marker, e.g., luciferase), or detecting a cellularresponse, for example, development, differentiation or rate ofproliferation.

In yet another embodiment, an assay of the present invention is acell-free assay in which a Dkk or Dkk-related protein or biologicallyactive portion thereof is contacted with a test compound and the abilityof the test compound to bind to the Dkk or Dkk-related protein orbiologically active portion thereof is determined. Binding of the testcompound to the Dkk or Dkk-related protein can be determined eitherdirectly or indirectly as described above. In a preferred embodiment,the assay includes contacting the Dkk or Dkk-related protein orbiologically active portion thereof with a known compound which bindsDkk or the Dkk-related protein to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a Dkk or Dkk-related protein, whereindetermining the ability of the test compound to interact with a Dkk orDkk-related protein comprises determining the ability of the testcompound to preferentially bind to Dkk or a Dkk-related protein orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a Dkk orDkk-related protein or biologically active portion thereof is contactedwith a test compound and the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the Dkk or Dkk-relatedprotein or biologically active portion thereof is determined.Determining the ability of the test compound to modulate the activity ofa Dkk or Dkk-related protein can be accomplished, for example, bydetermining the ability of the Dkk or Dkk-related protein to bind to atarget molecule (e.g., a Dkk-target molecule) by one of the methodsdescribed above for determining direct binding. Determining the abilityof the Dkk or Dkk-related protein to bind to a target molecule can alsobe accomplished using a technology such as real-time BiomolocularInteraction Analysis (BIA). Sjolander, S, and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore™). Changes in the optical phenomenon surfaceplasmon resonance (SPR) can be used as an indication of real-timereactions between biological molecules.

In an alternative embodiment. determining the ability of the testcompound to modulate the activity of a Dkk or Dkk-related protein can beaccomplished by determining the ability of the Dkk or Dkk-relatedprotein to further modulate the activity of a target molecule (e.g., aDkk-target molecule). For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined aspreviously described.

In yet another embodiment, the cell-free assay involves contacting a Dkkor Dkk-related protein or biologically active portion thereof with aknown compound which binds the Dkk or Dkk-related protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with the Dickor Dkk-related protein, wherein determining the ability of the testcompound to interact with the Dkk or Dkk-related protein comprisesdetermining the ability of the Dkk or Dkk-related protein topreferentially bind to or modulate the activity of a target molecule(e.g., a Dkk target molecule).

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, the compound of interest is contacted with a Dkk(e.g., hDkk-3) protein or a Dkk (e.g., hDkk-3) binding partner, e.g., areceptor. The receptor can be soluble or the receptor can be present ona cell surface. To the mixture of the compound and the Dkk protein orDkk binding partner is then added a composition containing a Dkk bindingpartner or a Dkk protein, respectively. Detection and quantification ofcomplexes of Dkk proteins and Dkk binding partners provide a means fordetermining a compound's efficacy at inhibiting (or potentiating)complex formation between Dkk and a binding partner. The efficacy of thecompound can be assessed by generating dose response curves from dataobtained using various concentrations of the test compound. Moreover, acontrol assay can also be performed to provide a baseline forcomparison. In the control assay, isolated and purified Dkk polypeptideor binding partner is added to a composition containing the Dkk bindingpartner or Dkk polypeptide, and the formation of a complex isquantitated in the absence of the test compound.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins (e.g. Dkkproteins or biologically active portions thereof or Dkk targetmolecules), in the case of cell-free assays in which a membrane-boundform an isolated protein is used (e.g., a Dkk target molecule orreceptor) it may be desirable to utilize a solubilizing agent such thatthe membrane-bound form of the isolated protein is maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either Dkk, a Dkk-relatedprotein or a target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a Dkkor Dkk-related protein, or interaction of a Dkk or Dkk-related proteinwith a target molecule in the presence and absence of a candidatecompound, can be accomplished in any vessel suitable for containing thereactants. Examples of such vessels include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows one or both of theproteins to be bound to a matrix. For example,glutathione-S-transferase/Dkk fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis. MO) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or Dkk protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of Dkkbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a Dkkprotein, Dkk-related protein, or a Dkk target molecule can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated protein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical),Alternatively, antibodies reactive with Dkk, Dkk-related protein, ortarget molecules but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andunbound target, Dkk, or Dkk-related protein trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the Dkk orDkk-related protein or target molecule, as well as enzyme-linked assayswhich rely on detecting an enzymatic activity associated with the Dkk orDkk-related protein or target molecule.

In another embodiment, modulators of Dkk or Dkk-related expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of Dkk or Dkk-related mRNA or protein in thecell is determined. The level of expression of mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of mRNA or protein in the absence of the candidate compound.The candidate compound can then be identified as a modulator of Dkk orDkk-related expression based on this comparison. For example, whenexpression of Dkk mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator of DkkmRNA or protein expression. Alternatively, when expression of Dkk mRNAor protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of Dkk mRNA or protein expression. The levelof Dkk or Dkk-related mRNA or protein expression in the cells can bedetermined by methods described herein for detecting Dkk mRNA orprotein.

In yet another aspect of the invention, the Dkk or Dkk-related proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al., (1993) Biotechniques 14:920-934; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with Dkk or Dkk-related proteins (“binding proteins”or and modulate Dkk or Dkk-related activity. Such binding proteins arealso likely to be involved in the propagation of signals by the Dkk orDkk-related proteins as, for example, downstream elements of aDkk-mediated signaling pathway. Alternatively, such binding proteins arelikely to be cell-surface molecules associated with non-Dkk expressingcells, wherein such binding proteins are involved in signaltransduction.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a Dick protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence. from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a Dkk-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the Dkk orDkk-related protein.

This invention further pertains to novel agents identified by theabove-described screening assays and to processes for producing suchagents by use of these assays. Accordingly. in one embodiment, thepresent invention includes a compound or agent obtainable by a methodcomprising the steps of any one of the aformentioned screening assays(e.g., cell-based assays or cell-free assays). For example, in oneembodiment. the invention includes a compound or agent obtainable by amethod comprising contacting a cell which expresses a target moleculewith a test compound and the determining the ability of the testcompound to bind to, or modulate the activity of, the target molecule.In another embodiment, the invention includes a compound or agentobtainable by a method comprising contacting a cell which expresses atarget molecule with a Dkk or Dkk-related protein or biologically-activeportion thereof, to form an assay mixture. contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with, or modulate the activity of, the target molecule. Inanother embodiment, the invention includes a compound or agentobtainable by a method comprising contacting a Dkk or Dkk-relatedprotein or biologically active portion thereof with a test compound anddetermining the ability of the test compound to bind to, or modulate(e.g., stimulate or inhibit) the activity of. the Dkk or Dkk-relatedprotein or biologically active portion thereof. In yet anotherembodiment, the present invention includes a compound or agentobtainable by a method comprising contacting a Dkk or Dkk-relatedprotein or biologically active portion thereof with a known compoundwhich binds the Dkk or Dkk-related protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with, or modulate the activityof the Dkk or Dkk-related protein.

Accordingly, it is within the scope of this invention to further use anagent identified as described herein in an appropriate animal model. Forexample. an agent identified as described herein (e.g., a Dkk modulatingagent, an antisense Dkk nucleic acid molecule, a Dkk-specific antibody,or a Dkk-binding partner) can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.

The present invention also pertains to uses of novel agents identifiedby the above-described screening assays for diagnoses, prognoses, andtreatments as described herein. Accordingly, it is within the scope ofthe present invention to use such agents in the design, formulation,synthesis, manufacture, and/or production of a drug or pharmaceuticalcomposition for use in diagnosis, prognosis, or treatment, as describedherein. For example, in one embodiment, the present invention includes amethod of synthesizing or producing a drug or pharmaceutical compositionby reference to the structure and/or properties of a compound obtainableby one of the above-described screening assays. For example, a drug orpharmaceutical composition can be synthesized based on the structureand/or properties of a compound obtained by a method in which a cellwhich expresses a target molecule (e.g., a Dkk target molecule) iscontacted with a test compound and the ability of the test compound tobind to, or modulate the activity of, the target molecule is determined.In another exemplary embodiment, the present invention includes a methodof synthesizing or producing a drug or pharmaceutical composition basedon the structure and/or properties of a compound obtainable by a methodin which a Dkk or Dkk-related protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to, or modulate (e.g., stimulate or inhibit) theactivity of, the Dkk or Dkk-related protein or biologically activeportion thereof is determined.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the Dkk or Dkk-related nucleotide sequences,described herein, can be used to map the location of the Dkk orDkk-related genes on a chromosome. The mapping of the Dkk or Dkk-relatedsequences to chromosomes is an important first step in correlating thesesequences genes associated with disease.

Briefly, Dkk or Dkk-related genes can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 by in length) from the Dkk orDkk-related nucleotide sequences. Computer analysis of the Dkk orDkk-related sequences can be used to predict primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the Dkk orDkk-related sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media. panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al., (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the Dkk orDkk-related nucleotide sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa 9o, 1p, or 1v sequence to its chromosome include in situ hybridization(described in Fan, Y. et al., (1990) PNAS, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with a Dkk or Dkk-related gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

2. Tissue Typing

The Dkk or Dkk-related sequences of the present invention can also beused to identify individuals from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique. an individual's genomic DNA is digested with one ormore restriction enzymes. and probed on a Southern blot to yield uniquebands for identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the Dkk or Dkk-related nucleotide sequences describedherein can be used to prepare two PCR primers from the 5′ and 3′ ends ofthe sequences. These primers can then be used to amplify an individual'sDNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner. can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The Dkk or Dkk-related nucleotide sequences of the invention uniquelyrepresent portions of the human genome. Allelic variation occurs to somedegree in the coding regions of these sequences, and to a greater degreein the noncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, SEQID NO:4. SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3. SEQID NO:6, SEQ ID NO:9. SEQ ID NO:15 or SEQ ID NO:22 are used, a moreappropriate number of primers for positive individual identificationwould be 500-2,000.

If a panel of reagents from Dkk or Dkk-related nucleotide sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

3. Use of Partial Dkk or Dkk-related Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example. providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NOs:1, SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:13, or SEQ ID NO:20 are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the Dkk nucleotide sequencesor portions thereof, e.g., fragments derived from the noncoding regionsof SEQ ID NO:1, SEQ ID NO:4. SEQ ID NO:7, SEQ ID NO:13. or SEQ ID NO:20.having a length of at least 20 bases, preferably at least 30 bases.

The Dkk or Dkk-related nucleotide sequences described herein can furtherbe used to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue, e.g., brain tissue. This canbe very useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such Dkk or Dkk-related probes canbe used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents. e.g., Dkk or Dkk-related primersor probes can be used to screen tissue culture for contamination (i.e.screen for the presence of a mixture of different types of cells in aculture).

C. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining Dkk orDkk-related protein and/or nucleic acid expression as well as Dkk orDkk-related activity, in the context of a biological sample (e.g.,blood, serum, cells, tissue) to thereby determine whether an individualis afflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant Dkk or Dkk-related expression oractivity, such as aberrant cell proliferation, differentiation. and/orsurvival resulting for example in a neurodegenerative disease (e.g.,Alzheimer's disease, Parkinson's disease, Huntington's chorea,amylotrophic lateral sclerosis and the like, as well as spinocerebellardegenerations) or cancer (for example, cancers of the epithelia (e.g.,carcinomas of the pancreas, stomach, liver, secretory glands (e.g.,adenocarcinoma) bladder, lung, breast, skin (e.g., malignant melanoma),reproductive tract including prostate gland, ovary, cervix and uterus);cancers of the hematopoietic and immune system (e.g., leukemias andlymphomas); cancers of the central nervous, brain system and eye (e.g.,gliomas, glioblastoma, neuroblastoma and retinoblastoma); and cancers ofconnective tissues, bone, muscles and vasculature (e.g., sarcomas)). Theinvention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with Dkk or Dkk-related protein, nucleic acid expression oractivity. For example, mutations in a Dkk or Dkk-related gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby phophylactically treat an individualprior to the onset of a disorder characterized by or associated with Dkkor Dkk-related protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents, (e.g., drugs, compounds) on the expression or activity of Dkk orDkk-related in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of Dkk orDkk-related protein or nucleic acid in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a compound or an agent capable of detecting Dkkor Dkk-related protein or nucleic acid (e.g., mRNA, genomic DNA) thatencodes Dkk or Dkk-related protein such that the presence of Dkk orDkk-related protein or nucleic acid is detected in the biologicalsample. A preferred agent for detecting Dkk or Dkk-related mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toDkk or Dkk-related mRNA or genomic DNA. The nucleic acid probe can be,for example, a full-length Dkk nucleic acid, such as the nucleic acid ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, theDNA insert of the plasmid deposited with ATCC as Accession Number 98457,the DNA insert of the plasmid deposited with ATCC as Accession Number98633, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 207140, or a portion thereof,such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to Dkk or Dkk-related mRNA or genomie DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

A preferred agent for detecting Dkk or Dkk-related protein is anantibody capable of binding to the protein, preferably an antibody witha detectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect Dkk orDkk-related mRNA. protein, or genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof Dkk or Dkk-related mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of Dkk or Dkk-relatedprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots. immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of Dkk or Dkk-related genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of Dkk orDkk-related protein include introducing into a subject a labeledantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting Dkk or Dkk-relatedprotein, mRNA. or genomic DNA, such that the presence of Dkk orDkk-related protein. mRNA or genomic DNA is detected in the biologicalsample, and comparing the presence of Dkk or Dkk-related protein. mRNAor genomic DNA in the control sample with the presence of Dkk orDkk-related protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of Dkk ora Dkk-related protein in a biological sample. For example, the kit cancomprise a labeled compound or agent capable of detecting Dkk orDkk-related protein or mRNA in a biological sample; means fordetermining the amount of Dkk or Dkk-related protein or mRNA in thesample; and means for comparing the amount of Dkk or Dkk-related proteinor mRNA in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect Dkk or Dkk-related protein ornucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant Dkk expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with Dkk or Dkk-relatedprotein, nucleic acid expression or activity such as a proliferativedisorder, a differentiative or developmental disorder, a hematopoieticdisorder as well as diseases, conditions or disorders characterized byabnormal cell survival, abnormal extracellular structure, or anabnormality in a defense mechanism. Alternatively, the prognostic assayscan be utilized to identify a subject having or at risk for developing adifferentiative or proliferative disease (e.g., cancer). Thus, thepresent invention provides a method for identifying a disease ordisorder associated with aberrant Dkk or Dkk-related expression oractivity in which a test sample is obtained from a subject and Dkk orDkk-related protein or nucleic acid (e.g., mRNA, genomic DNA) isdetected, wherein the presence of Dkk or Dkk-related protein or nucleicacid is diagnostic for a subject having or at risk of developing adisease or disorder associated with aberrant Dkk or Dkk-relatedexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example. atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant Dkk or Dkk-related expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a disorder, such as aproliferative disorder, a differentiative or developmental disorder, ahematopoietic disorder, as well disorders characterized by abnormal cellsurvival, an abnormal extracellular structure, or an abnormality in adefense mechanism. Alternatively, such methods can be used to determinewhether a subject can be effectively treated with an agent for adifferentiative or proliferative disease (e.g., cancer). Thus, thepresent invention provides methods for determining whether a subject canbe effectively treated with an agent for a disorder associated withaberrant Dkk or Dkk-related expression or activity in which a testsample is obtained and Dkk or Dkk-related protein or nucleic acidexpression or activity is detected (e.g., wherein the abundance of Dkkor Dkk-related protein or nucleic acid expression or activity isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant Dkk or Dkk-related expression oractivity.)

The methods of the invention can also be used to detect geneticalterations in a Dkk or Dkk-related gene, thereby determining if asubject with the altered gene is at risk for a disorder characterized byaberrant development, aberrant cellular differentiation, aberrantcellular proliferation or an aberrant hematopoietic response. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a Dkk or Dkk-related-protein, or the mis-expressionof the Dkk or Dkk-related gene. For example, such genetic alterationscan he detected by ascertaining the existence of at least one of 1) adeletion of one or more nucleotides from a Dkk or Dkk-related gene; 2)an addition of one or more nucleotides to a Dkk or Dkk-related gene; 3)a substitution of one or more nucleotides of a Dkk or Dkk-related gene,4) a chromosomal rearrangement of a Dkk or Dkk-related gene; 5) analteration in the level of a messenger RNA transcript of a Dkk orDkk-related gene, 6) aberrant modification of a Dkk or Dkk-related gene.such as of the methylation pattern of the genomic DNA, 7) the presenceof a non-wild type splicing pattern of a messenger RNA transcript of aDkk or Dkk-related gene, 8) a non-wild type level of a Dkk orDkk-related-protein, 9) allelic loss of a Dkk or Dkk-related gene, and10) inappropriate post-translational modification of a Dkk orDkk-related-protein. As described herein, there are a large number ofassay techniques known in the art which can be used for detectingalterations in a Dkk or Dkk-related gene. A preferred biological sampleis a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:10477-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the Dkk or Dkk-related-gene (see Abravayaet al. (1995) Nucleic Acids Res. 23:675-682). This method can includethe steps of collecting a sample of cells from a patient, isolatingnucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a Dkk or Dkk-related gene under conditionssuch that hybridization and amplification of the Dkk or Dkk-related-gene(if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh. D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988. Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a Dkk or Dkk-related genefrom a sample cell can be identified by alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by getelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in a Dkk or Dkk-related gene canbe identified by hybridizing a sample and control nucleic acids, e.g.,DNA or RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin, M, T. et al. (1996) Human Mutation7:244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:753-759). Forexample, genetic mutations in Dkk can be identified in two dimensionalarrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra. Briefly, a first hybridization array of probes can beused to scan through long stretches of DNA in a sample and control toidentify base changes between the sequences by making linear arrays ofsequential ovelapping probes. This step allows the identification ofpoint mutations. This step is followed by a second hybridization arraythat allows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Dkk or Dkk-relatedgene and detect mutations by comparing the sequence of the sample Dkk orDkk-related sequence with the corresponding wild-type (control)sequence, Examples of sequencing reactions include those based ontechniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger((1977) PNAS 74:5463). It is also contemplated that any of a variety ofautomated sequencing procedures can be utilized when performing thediagnostic assays ((1995) Biotechniques 19:448), including sequencing bymass spectrometry (see, e.g., PCT International Publication No. WO94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162: and Griffinet al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the Dkk or Dkk-related geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type Dkk or Dkk-relatedsequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to basepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment. thecontrol DNA or RNA can be labeled for detection.

In still another embodiment” the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Dkk cDNAs obtained from samplesof cells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves TatG/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Dkk sequence,e.g., a wild-type Dkk sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in Dkk or Dkk-related genes. For example,single strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci. USA:86:2766,see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79). Single-stranded DNA fragments of sample andcontrol Dkk or Dkk-related nucleic acids will be denatured and allowedto renature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment” the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 by of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:17753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Dkk gene.

Furthermore, any cell type or tissue in which Dkk or a Dkk-relatedsequence is expressed may be utilized in the prognostic assays describedherein.

3. Monitoring of Effects Durine Clinical Trials

Monitoring the influence of agents (e.g., drugs. compounds) on theexpression or activity of Dkk or Dkk-related molecule (e.g., modulationof cellular signal transduction, regulation of gene transcription in acell involved in development or differentiation, regulation of cellularproliferation) can be applied not only in basic drug screening, but alsoin clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase Dkk orDkk-related gene expression. protein levels, or upregulate Dkk orDkk-related activity, can be monitored in clinical trials of subjectsexhibiting decreased Dkk or Dkk-related gene expression, protein levels,or downregulated Dkk or Dkk-related activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreaseDkk or Dkk-related gene expression, protein levels, or downregulate Dkkor Dkk-related activity, can be monitored in clinical trials of subjectsexhibiting increased Dkk or Dkk-related gene expression, protein levels,or upregulated Dkk or Dkk-related activity. In such clinical trials, theexpression or activity of Dkk or Dkk-related and, preferably, othergenes that have been implicated in, for example, a proliferativedisorder can be used as a “read out” or markers of the phenotype of aparticular cell.

For example, and not by way of limitation, genes, including Dkk andDkk-related genes, that are modulated in cells by treatment with anagent (e.g., compound, drug or small molecule) which modulates Dkk orDkk-related activity (e.g., identified in a screening assay as describedherein) can be identified. Thus, to study the effect of agents onproliferative disorders, developmental or differentiative disorder,hematopoietic disorder as well disorders characterized by abnormal celldifferentiation and/or survival, an abnormal extracellular structure, oran abnormality in a defense mechanism, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of Dkk, Dkk-related, and other genes implicated in theproliferative disorder, developmental or differentiative disorder,hematopoietic disorder as well as disorders characterized by abnormalcell differentiation and/or survival, an abnormal extracellularstructure, or an abnormality in a defense mechanism, respectively. Thelevels of gene expression (i.e., a gene expression pattern) can hequantified by Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofDkk, Dkk-related, or other genes. In this way, the gene expressionpattern can serve as a marker, indicative of the physiological responseof the cells to the agent. Accordingly, this response state may hedetermined before, and at various points during treatment of theindividual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist. antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a Dkk orDkk-related protein, mRNA, or genomic DNA in the preadministrationsample; (iii) obtaining one or more post administration samples from thesubject; (iv) detecting the level of expression or activity of the Dkkor Dkk-related protein, mRNA, or genomic DNA in the post-administrationsamples; (v) comparing the level of expression or activity of the Dkk orDkk-related protein, mRNA, or genomic DNA in the pre-administrationsample with the Dkk or Dkk-related protein, mRNA, or genomic DNA in thepost administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of Dkk or Dkk-related nucleic acid or protein tohigher levels than detected, i.e., to increase the effectiveness of theagent. Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of Dkk or Dkk-relatednucleic acid or protein to lower levels than detected, i.e. to decreasethe effectiveness of the agent. According to such an embodiment, Dkk orDkk-related expression or activity may be used as an indicator of theeffectiveness of an agent, even in the absence of an observablephenotypic response.

C. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant Dkk or Dkk-relatedexpression or activity. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. “Pharmacogenomics”, as used herein, refers to theapplication of genomics technologies such as gene sequencing,statistical genetics, and gene expression analysis to drugs in clinicaldevelopment and on the market. More specifically, the term refers thestudy of how a patient's genes determine his or her response to a drug(e.g., a patient's “drug response phenotype”, or “drug responsegenotype”.) Thus, another aspect of the invention provides methods fortailoring an individual's prophylactic or therapeutic treatment witheither the Dkk or Dkk-related molecules of the present invention or Dkkor Dkk-related modulators according to that individual's drug responsegenotype. Pharmacogenomics allows a clinician or physician to targetprophylactic or therapeutic treatments to patients who will most benefitfrom the treatment and to avoid treatment of patients who willexperience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant Dkk orDkk-related expression or activity, by administering to the subject anagent which modulates Dkk or Dkk-related expression or at least one Dkkor Dkk-related activity. Subjects at risk for a disease which is causedor contributed to by aberrant Dkk or Dkk-related expression or activitycan be identified by. for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe Dkk or Dkk-related aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of Dkk or Dkk-related aberrancy, for example, an agonist orantagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein. Theprophylactic methods of the present invention are further discussed inthe following subsections.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating Dkk orDkk-related expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of Dkk or Dkk-relatedprotein activity associated with the cell. An agent that modulates Dkkor Dkk-related protein activity can be an agent as described herein,such as a nucleic acid or a protein, a naturally-occurring targetmolecule of a Dkk or Dkk-related protein, a peptide, a Dkk orDkk-related peptidomimetic, or other small molecule. In one embodiment,the agent stimulates one or more Dkk or Dkk-related protein activity.Examples of such stimulatory agents include active Dkk or Dkk-relatedprotein and a nucleic acid molecule encoding Dkk or Dkk-related that hasbeen introduced into the cell. In another embodiment, the agent inhibitsone or more Dkk or Dkk-related protein activity. Examples of suchinhibitory agents include antisense Dkk or Dkk-related nucleic acidmolecules and antibodies. These modulatory methods can be performed invitro (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g., by administering the agent to a subject). As such, thepresent invention provides methods of treating an individual afflictedwith a disease or disorder characterized by aberrant expression oractivity of a Dkk or Dkk-related protein or nucleic acid molecule. Thepresent invention also provides methods of modulating the function,morphology. proliferation, and/or differentiation of cells in thetissues in which a Dkk or Dkk-related protein or nucleic acid moleculeis expressed. Alternatively, Dkk or Dkk-related polypeptides, nucleicacids, and modulators thereof, can be used to treat disorders associatedwith abnormal or aberrant metabolism or function of cells in the tissuesin which the Dkk or Dkk-related protein or nucleic acid molecule isexpressed.

For example, tissues in which Dkk-3 is expressed include embryonic eye,bone, and cartilage, fetal brain, lung, and kidney, and adult heart (inparticular, atrioventricular valves and atrial myocytes), eye inparticular, the integrating bipolar and ganglion cells of the retina,the ciliary body, and lens epithelium), brain (in particular, neurons ofthe cortex and hippocampus), placenta, lung, and skeletal muscle.Accordingly, Dkk-3 polypeptides. nucleic acids, or modulators thereof,can be used to treat cardiovascular disorders, such as ischemic heartdisease (e.g., angina pectoris, myocardial infarction, and chronicischemic heart disease), hypertensive heart disease, pulmonary heartdisease, valvular heart disease (e.g., rheumatic fever and rheumaticheart disease, endocarditis, mitral valve prolapse, and aortic valvestenosis), congenital heart disease (e.g., valvular and vascularobstructive lesions, atrial or ventricular septal defect, and patentductus arteriosus), or myocardial disease (e.g., myocarditis, congestivecardiomyopathy, and hypertrophic cariomyopathy).

In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat optic disorders such as diseasesassociated with amaurosis (e.g., a. fugax and a. albuminuric) diseasesassociated with amblyopia, glaucoma, optic neuropathy (e.g., ischemicneuropathy, optic neuritis, and infiltrative neuropathy), opthalmia(e.g., o. catarrhal, trachoma, o. neuroparalytic, and conjunctiva),visual disorders resulting from systemic disease or disorders of othertissues (e.g., diabetes mellitus, hyperthyroidism, and vitamin A orriboflavin deficiency), or tumors, neoplasms, and metastases.

In another embodiment. Dkk-3 polypeptides. nucleic acids, or modulatorsthereof, can be used to treat disorders of the brain, such as cerebraledema, senile dementia of the Alzeimer type, epilepsy, amnesia,hydrocephalus, brain herniations, iatrogenic disease (due to, e.g.,infection, toxins, or drugs), inflammations (e.g., bacterial and viralmeningitis, encephalitis, and cerebral toxoplasmosis), cerebrovasculardiseases (e.g., hypoxia, ischemia, and infarction, intracranialhemorrhage and vascular malformations, and hypertensive encephalopathy),and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pinealcells, meningeal tumors, primary and secondary lymphomas, intracranialtumors, and medulloblastoma), and to treat injury or trauma to thebrain.

In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat placental disorders, such as toxemia ofpregnancy (e.g., preeclampsia and eclampsia), placentitis, orspontaneous abortion.

In another embodiment, Dkk-3 polypeptides. nucleic acids, or modulatorsthereof, can be used to treat pulmonary disorders, such as atelectasis,pulmonary congestion or edema, chronic obstructive airway disease (e.g.,emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis),diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis,hypersensitivity pneumonitis. Goodpasture's syndrome, idiopathicpulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamativeinterstitial pneumonitis, chronic interstitial pneumonia, fibrosingalveolitis, hammanrich syndrome, pulmonary eosinophilia, diffuseinterstitial fibrosis, Wegener's granulomatosis, lymphomatoidgranulomatosis, and lipid pneumonia), or tumors (e.g., bronchogeniccarcinoma, bronchioloalveolar carcinoma, bronchial carcinoid, hamartoma,and mesenchymal tumors).

In another embodiment. Dkk-3 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat disorders of skeletal muscle, such asmuscular atrophy (due to, e.g., denervation, malnutrition, loss of bloodsupply, or neuromuscular disease. e.g., amyotonia congenita, amyotrophiclateral sclerosis of Charcot, and progressive muscular atrophy ofAran-Duchenne), myositis (due to, e.g., bacterial, viral, fungal orparasitic infection), muscular dystrophies (e.g., Duchenne type, Beckertype, facioscapulohumeral, limb-girdle, myotonic dystrophy, and ocularmyopathy), myasthenia gravis, or tumors and tumor-like lesions ofmuscles (e.g., traumatic myositis ossificans, desmoids,musculoaponeurotic fibromatosis, Dupuytren's contracture, nodular(pseudosarcomatous) fasciitis, rhadomyoma, rhabdomyosarcoma, andgranular cell myoblastomas).

Tissues in which Dkk-4 is expressed include cerebellum, activated humanT-lymphocytes, lung, and esophagus. Accordingly, in one embodiment,Dkk-4 polypeptides, nucleic acids, or modulators thereof, can be used totreat disorders of the cerebellum, such as disturbances of synergy(e.g., asynergia or limb ataxia, dysmetria, decomposition of movement,hypermetria, hypometria, dysdiadochokinesia, hypotonia, tremor,dysarthria, nystagmus), disturbances of equilibrium (due to, e.g., alesion involving the vestibulocerebellum), disturbances of gait stance,or tone (due to, e.g., a lesion or degeneration of the spinocerebellum),or tumors (e.g., astrocytoma and medulloblastoma).

In another embodiment, Dkk-4 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat lymphocytic disorders, such aslymphopenia, lymphocytosis, acute and chronic lymphadenitis, malignantlymphomas (e.g., Non-Hodgkin's lymphomas, Hodgkin's lymphomas,leukemias, multiple myeloma, histiocytoses, and angioimmunoblasticlymphadenopathy).

In another embodiment. Dkk-4 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat pulmonary disorders, such as atelectasis,pulmonary congestion or edema, chronic obstructive airway disease (e.g.,emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis),diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis,hypersensitivity pneumonitis. Goodpasture's syndrome, idiopathicpulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamativeinterstitial pneumonitis, chronic interstitial pneumonia, fibrosingalveolitis, hammanrich syndrome, pulmonary eosinophilia, diffuseinterstitial fibrosis, Wegener's granulomatosis, lymphomatoidgranulomatosis, and lipid pneumonia), or tumors (e.g., bronchogeniccarcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid, hamartoma,and mesenchymal tumors).

In another embodiment, Dkk-4 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat esophageal disorders, such asneuromuscular disturbances (e.g., achalasia, annular narrowings,Schatzki's rings, hiatal hernia, Mallory-Weiss syndrome), esophagitis(due to e.g., bacteremia, viremia, fungal infections, uremia,graft-versus-host disease, chemotherapy, radiation, and prolongedgastric intubation), diverticula (e.g., Zenker's diverticulum), systemicsclerosis, varices (due to, e.g., portal hypertension, systemicamyloidosis and sarcoidosis), or tumors or neoplasms (e.g., leimyoma,fibromas, lipomas, hemangiomas, lymphangiomas, squamous papillomas,adenocarcinomas and undifferentiated carcinomas, and sarcomas).

Dkk-1 is highly expressed, for example, in placenta. Accordingly, Dkk-1polypeptides, nucleic acids, or modulators thereof, can be used to treatplacental disorders, such as toxemia of pregnancy (e.g., preeclampsiaand eclampsia), placentitis, or spontaneous abortion.

Tissues in which Dkk-2 is expressed include, for example, heart, brain,placenta, lung, and skeletal muscle. Accordingly, Dkk-2 polypeptides,nucleic acids, or modulators thereof, can be used to treatcardiovascular disorders, such as ischemic heart disease (e.g., anginapectoris, myocardial infarction, and chronic ischemic heart disease),hypertensive heart disease, pulmonary heart disease, valvular heartdisease (e.g., rheumatic fever and rheumatic heart disease,endocarditis, mitral valve prolapse, and aortic valve stenosis),congenital heart disease (e.g., valvular and vascular obstructivelesions, atrial or ventricular septal defect, and patent ductusarteriosus), or myocardial disease (e.g., myocarditis, congestivecardiomyopathy, and hypertrophic cariomyopathy).

In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat disorders of the brain, such as cerebraledema, senile dementia of the Alzeimer type, epilepsy, amnesia,hydrocephalus, brain herniations, iatrogenic disease (due to, e.g.,infection, toxins, or drugs), inflammations (e.g., bacterial and viralmeningitis, encephalitis, and cerebral toxoplasmosis), cerebrovasculardiseases (e.g., hypoxia, ischemia, and infarction, intracranialhemorrhage and vascular malformations, and hypertensive encephalopathy),and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pinealcells, meningeal tumors, primary and secondary lymphomas, intracranialtumors, and medulloblastoma), and to treat injury or trauma to thebrain.

In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat placental disorders, such as toxemia ofpregnancy (e.g., preeclampsia and eclampsia), placentitis, orspontaneous abortion.

In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat pulmonary disorders, such as atelectasis,pulmonary congestion or edema, chronic obstructive airway disease (e.g.,emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis),diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis,hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathicpulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamativeinterstitial pneumonitis, chronic interstitial pneumonia, fibrosingalveolitis, hammanrich syndrome, pulmonary eosinophilia, diffuseinterstitial fibrosis, Wegener's granulomatosis, lymphomatoidgranulomatosis, and lipid pneumonia), or tumors (e.g., bronchogeniccarcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid, hamartoma,and mesenchymal tumors).

In another embodiment. Dkk-2 polypeptides, nucleic acids, or modulatorsthereof, can be used to treat disorders of skeletal muscle, such asmuscular atrophy (due to, e.g., denervation, malnutrition, loss of bloodsupply, or neuromuscular disease, e.g., amyotonia congenita, amyotrophiclateral sclerosis of Charcot, and progressive muscular atrophy ofAran-Duchenne), myositis (due to, e.g., bacterial, viral, fungal orparasitic infection), muscular dystrophies (e.g., Duchenne type, Beckertype, facioscapulohumeral, limb-girdle, myotonic dystrophy, and ocularmyopathy), myasthenia gravis, or tumors and tumor-like lesions ofmuscles (e.g., traumatic myositis ossificans, desmoids,musculoaponeurotic fibromatosis, Dupuytren's contracture, nodular(pseudosarcomatous) fasciitis, rhadomyoma, rhabdomyosarcoma, andgranular cell myoblastomas).

Soggy-1 is expressed in, for example, testis (e.g., spermatogenicepithelium of the seminiferous tubules, spermatogonia) and in embryonicdeveloping dorsal root ganglia, cartilage primordium of the nasalseptum, and the eye. Accordingly, Soggy-1 polypeptides, nucleic acids,or modulators thereof, can be used to treat testicular disorders, suchas unilateral testicular enlargement (e.g., nontuberculous,granulomatous orchitis), inflammatory diseases resulting in testiculardysfunction (e.g., gonorrhea and mumps), and tumors (e.g., germ celltumors, interstitial cell tumors, androblastoma, testicular lymphoma andadenomatoid tumors). In another embodiment, Soggy-1 polypeptides,nucleic acids, or modulators thereof, can be used to treat infertilitydue to, for example, spermatogenetic failure.

In one aspect, the above-described methods involve administering anagent (e.g., an agent identified by a screening assay described herein),or combination of agents that modulates (e.g., upregulates ordownregulates) Dkk or Dkk-related expression or activity. In anotherembodiment, the method involves administering a Dkk or Dkk-relatedprotein or nucleic acid molecule as therapy to compensate for reduced oraberrant Dkk or Dkk-related expression or activity.

A preferred embodiment of the present invention involves a method fortreatment of a disease or disorder associated with a Dkk or Dkk-relatedprotein which includes the step of administering a therapeuticallyeffective amount of an antibody to a Dkk or Dkk-related protein to asubject. As defined herein, a therapeutically effective amount ofantibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kgbody weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody in the range ofbetween about 0.1 to 20 mg/kg body weight, one time per week for betweenabout 1 to 10 weeks, preferably between 2 to 8 weeks, more preferablybetween about 3 to 7 weeks, and even more preferably for about 4, 5, or6 weeks. It will also be appreciated that the effective dosage ofantibody used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results ofdiagnostic assays as described herein.

Stimulation of Dkk or Dkk-related activity is desirable in situations inwhich Dkk or Dkk-related activity is abnormally downregulated and/or inwhich increased Dkk or Dkk-related activity is likely to have abeneficial effect. Likewise, inhibition of Dkk or Dkk-related activityis desirable in situations in which Dkk or Dkk-related activity isabnormally upregulated and/or in which decreased Dkk or Dkk-relatedactivity is likely to have a beneficial effect. One example of such asituation is where a subject has a disorder characterized by aberrantdevelopment or cellular differentiation. Another example of such asituation is where the subject has a proliferative disease (e.g.,cancer) or a neurogenerative disorder. Yet another example of such asituation is where it is desirable to achieve tissue regeneration in asubject (e.g., where a subject has undergone brain or spinal cord injuryand it is desirable to regenerate neuronal tissue in a regulatedmanner.)

Accordingly, in one embodiment, the disease is a disease characterizedby an abnormal cell proliferation, differentiation, and/or survival. Forexample. the disease can be a hyper- or hypoproliferative disease. Theinvention also provides methods for treating diseases characterized byan abnormal cell proliferation, differentiation, and/or survival in asubject, which are not characterized by an abnormal Dkk or Dkk-relatedactivity (e.g., hDkk-3 activity). In fact, since Dkk is likely to becapable of modulating the proliferative state of a cell (i.e., state ofproliferation, differentiation, and or survival of a cell). Dkk canregulate disease wherein the abnormal proliferative state of a cellresults from a defect other than an abnormal Dkk activity.

Hyperproliferative diseases can be treated with Dkk or Dkk-related(e.g., hDkk-3) therapeutics include neoplastic and hyperplasticdiseases, such as various forms of cancers and leukemias, andfibroproliferative disorders. Other hyperproliferative diseases that canbe treated or prevented with the subject Dkk or Dkk-related therapeutics(e.g. hDkk-3 therapeutics) include malignant conditions, premalignantconditions, and benign conditions. The condition to be treated orprevented can be a solid tumor, such as a tumor arising in an epithelialtissue. Accordingly, treatment of such a cancer could compriseadministration to the subject of a Dkk or Dkk-related therapeuticdecreasing the interaction of Dkk with a Dkk receptor. Other cancersthat can be treated or prevented with a Dkk or Dkk-related proteininclude cancers of the epithelia (e.g., carcinomas of the pancreas,kidney, stomach, colon, esophagus liver, secretory glands (e.g.,adenocarcinoma) bladder, lung, breast, skin (e.g., malignant melanoma,seminoma squamous adenocarcinoma), reproductive tract including prostategland, testis, ovary, cervix and uterus); cancers of the hematopoieticand immune system (e.g., leukemias and lymphomas); cancers of thecentral nervous, brain system and eye (e.g., malignant astrocytoma,gliomas, neuroblastoma and retinoblastoma); and cancers of connectivetissues, bone, heart, muscles and vasculature (e.g., sarcomas, forexample, osteosarcoma). Additional solid tumors within the scope of theinvention include those that can be found in a medical textbook.

The condition to be treated or prevented can also be a soluble tumor,such as leukemia, either chronic or acute, including chronic or acutemyelogenous leukemia, chronic or acute lymphocytic leukemia,promyelocytic leukemia, monocytic leukemia, myelomonocytic leukemia, anderythroleukemia. Yet other proliferative disorders that can be treatedwith a Dkk or Dkk-related therapeutic of the invention include heavychain disease, multiple myeloma, lymphoma, e.g., Hodgkin's lymphoma andnon-Hodgkin's lymphoma, and Waldenstroem's macroglobulemia.

Diseases or conditions characterized b a solid or soluble tumor can betreated by administrating a Dkk or Dkk-related therapeutic eitherlocally or systemically, such that aberrant cell proliferation isinhibited or decreased. Methods for administering the compounds of theinvention are further described below.

The invention also provides methods for preventing the formation and/ordevelopment of tumors. For example, the development of a tumor can bepreceded by the presence of a specific lesion, such as a pre-neoplasticlesion, e.g., hyperplasia, metaplasia, and dysplasia, which can bedetected, e.g., by cytologic methods. Such lesions can be found, e.g.,in epithelial tissue. Thus, the invention provides a method forinhibiting progression of such a lesion into a neoplastic lesion,comprising administering to the subject having a preneoplastic lesion anamount of a Dkk or Dkk-related therapeutic sufficient to inhibitprogression of the preneoplastic lesion into a neoplastic lesion.

The invention also provides for methods for treating or preventingdiseases or conditions in which proliferation of cells is desired. Forexample, Dkk or Dkk-related therapeutics can be used to stimulate tissuerepair or wound healing, such as after surgery or to stimulate tissuehealing from burns. Other diseases in which proliferation of cells isdesired are hypoproliferative diseases, i.e., diseases characterized byan abnormally low proliferation of certain cells.

In yet another embodiment, the invention provides a method for treatingor preventing diseases or conditions characterized by aberrant celldifferentiation. Accordingly, the invention provides methods forstimulating cellular differentiation in conditions characterized by aninhibition of normal cell differentiation which may or may not beaccompanied by excessive proliferation. Alternatively, Dkk orDkk-related therapeutics can be used to inhibit differentiation ofspecific cells.

In a preferred method, the aberrantly proliferating and/ordifferentiating cell is a cell present in the nervous system. A role forDkk in the nervous system is suggested at least in part from the factthat human Dkk-3 is expressed in human fetal brain. Accordingly, theinvention provides methods for treating diseases or conditionsassociated with a central or peripheral nervous system. For example, theinvention provides methods for treating lesions of the nervous systemassociated with an aberrant proliferation, differentiation or survivalof any of the following cells: cells of the central nervous systemincluding neurons and glial cells (e.g., astrocytes andoligodendrocytes) and supporting cells of peripheral neurons (e.g.,Schwann cells and satellite cells). Disorders of the nervous systeminclude, but are not limited to: spinal cord injuries, brain injuries,brain tumors (e.g., astrocytic tumors, for example, astrocytomas andglioblastomas), lesions associated with surgery, ischemic lesions,malignant lesions, infectious lesions, degenerative lesions (e.g.,Parkinson's disease, Alzheimer's disease, Huntington's chorea,amyotrophic lateral sclerosis), demyelinlating diseases (e.g., multiplesclerosis, human immunodeficiency associated myelopathy, transversemyelopathy, progressive multifocal leukoencephalopathy, pontinemyelinolysis), motor neuron injuries, progressive spinal muscularatrophy. progressive bulbar palsy, primary lateral sclerosis, infantileand juvenile muscular atrophy, progressive bulbar paralysis of childhood(i.e., Fazio-Londe syndrome), poliomyelitis, and hereditary motorsensoryneuropathy (i.e., Charcot-Marie-Tooth disease).

In another embodiment. the invention provides a method for enhancing thesurvival and/or stimulating proliferation and/or differentiation ofcells and tissues in vitro. In a preferred embodiment, Dkk orDkk-related therapeutics are used to promote tissue regeneration and/orrepair (e.g., to treat nerve injury). For example, tissues from asubject can be obtained and grown in vitro in the presence of a Dkk orDkk-related therapeutic, such that the tissue cells are stimulated toproliferate and/or differentiate. The tissue can then be readministeredto the subject.

Among the approaches which may be used to ameliorate disease symptomsinvolving an aberrant Dick or Dkk-related activity and/or an abnormalcell proliferation, differentiation, and/or survival, are, for example,antisense, ribozyme, and triple helix molecules described above.Examples of suitable compounds include the antagonists, agonists orhomologues described in detail above.

Yet other Dkk or Dkk-related therapeutics consist of a first peptidecomprising a Dkk or Dkk-related peptide capable of binding to a Dkkreceptor, and a second peptide which is cytotoxic. Such therapeutics canbe used to specifically target and lyse cells expressing oroverexpressing a receptor for Dkk.

3. Pharmacogenomics

The Dkk or Dkk-related molecules of the present invention, as well asagents, or modulators which have a stimulatory or inhibitory effect onDkk or Dkk-related activity (e.g., Dkk or Dkk-related gene expression)as identified by a screening assay described herein can be administeredto individuals to treat (prophylactically or therapeutically) disorders(e.g., proliferative or developmental disorders) associated withaberrant Dkk or Dkk-related activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a Dkk or Dkk-relatedmolecule or Dkk or Dkk-related modulator as well as tailoring the dosageand/or therapeutic regimen of treatment with a Dkk or Dkk-relatedmolecule or Dkk or Dkk-related modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See e.g., Eichelbaum, M., Clin Exp PharmacolPlysiol, 1996, 23(10-11): 983:-985 and Linder, M. W., Clin Chem. 1997,43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare genetic defects or as naturally-occurringpolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a gene marker map which consists of60.000-100,000 polymorphic or variable sites on the human genome, eachof which has two variants.) Such a high-resolution genetic map can becompared to a map of the genome of each of a statistically significantnumber of patients taking part in a Phase II/III drug trial to identifymarkers associated with a particular observed drug response or sideeffect. Alternatively, such a high resolution map can be generated froma combination of some ten-million known single nucleotide polymorphisms(SNPs) in the human genome. As used herein, a “SNP” is a commonalteration that occurs in a single nucleotide base in a stretch of DNA.For example, a SNP may occur once per every 1000 bases of DNA. A SNP maybe involved in a disease process, however, the vast majority may not bedisease-associated. Given a genetic map based on the occurrence of suchSNPs, individuals can be grouped into genetic categories depending on aparticular pattern of SNPs in their individual genome. In such a manner,treatment regimens can be tailored to groups of genetically similarindividuals, taking into account traits that may be common among suchgenetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a Dkkprotein or Dkk receptor of the present invention), all common variantsof that gene can be fairly easily identified in the population and itcan be determined if having one version of the gene versus another isassociated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a Dkk molecule orDkk modulator of the present invention) can give an indication whethergene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a Dkk molecule orDkk or Dkk-related modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references.patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

EXAMPLES

The invention is based, at least in part, on the discovery of a familyof genes encoding human cysteine-rich secreted proteins which arerelated to Xenopus Dickkopf (Dkk) proteins. This family includes hDkk-1,hDkk-2, hDkk-3, and hDkk-4, hDkks 1-4 contain two highly conservedcysteine-rich domains (CRDs), the most C-terminal of which demonstratessimilarity to the colipase protein family. The invention is based alsoin part on the discovery of a family of Dkk-related proteins, referredto as Soggy proteins, as well as the genes encoding Soggy proteins.Soggy-1 is a novel secreted protein which is related to the N-terminalregion of Dkk-3 but lacks CRDs. The following examples illustrate thestructure and function of each of these novel human secreted proteins.

Example 1 Isolation and Characterization of Human hDkk-3 cDNA

In this example, the isolation and characterization of the gene encodinghuman Dkk-3 (also referred to as “hDkk-3”, “Cysteine Rich SecretedProtein-1”, “CRSP-1” “CRISPY-1” or “TANGO 59”) is described.

Isolation of a Human Dkk-3 cDNA

The invention is based at least in part on the discovery of a human geneencoding a secreted protein, referred to herein as human Dickkopf-3(hDkk-3). A partial cDNA was isolated using a Signal Sequence Trapmethod. This methodology takes advantage of the fact that molecules suchas Dkk have an amino terminal signal sequence which directs certainsecreted and membrane-bound proteins through the cellular secretoryapparatus.

Briefly, a randomly primed cDNA library using mRNA prepared from humanfetal brain tissue (Clontech, Palo Alto Calif.) was made by using theStratagene-ZAP-cDNA Synthesis™ kit. (catalog #20041). The cDNA wasligated into the mammalian expression vector pTrap adjacent to a cDNAencoding placental alkaline phosphatase lacking a secretory signal. Theplasmids were transformed into E. coli and DNA was prepared using theWizard™ DNA purification kit (Promega). DNA was transfected into COS-7cells with Lipofectamine™ (Gibco-BRL). After 48 hours incubation the COScell supernatants were assayed for alkaline phosphatase on a WallacMicro-Beta scintillation counter using the Phospha-Light™ kit (TropixInc. Catalog #BP300). The individual plasmid DNAs scoring positive inthe COS cell Alkaline Phosphatase secretion assay were further analyzedby DNA sequencing using standard procedures.

Using a partial cDNA isolated by the above-described method (cloneAmhb3c3), a full length cDNA encoding human Dkk-3 was isolated from alambda Ziplox™ human fetal brain cDNA library using conventionalhybridization techniques (Sambrook et al., supra). The nucleotidesequence encoding the full length human Dkk-3 protein is shown in FIG.1A-1, 1A-2, 1B-1, 1B-2 and is set forth as SEQ ID NO:1. The full lengthprotein encoded by this nucleic acid is comprised of about 350 aminoacids and has the amino acid sequence shown in FIG. 1 and set forth asSEQ ID NO:2. The coding portion (open reading frame) of SEQ ID NO:1 isset forth as SEQ ID NO:3. DNA for the clone Fmhb059 was deposited withthe ATCC as Accession No. 98452.

Analysis of Human hDkk-3

Determination of the hydrophobicity profile of human Dkk-3 having theamino acid sequence set forth in SEQ ID NO:2 indicated the presence of ahydrophobic region from about amino acid 1 to about amino acid 22 of SEQID NO:2. Further analysis of the amino acid sequence SEQ ID NO:2 using asignal peptide prediction program predicted the presence of a signalpeptide from about amino acid 1 to about amino acid 22 of SEQ ID NO:2.Accordingly, the mature hDkk-3 protein includes about 328 amino acidsspanning from about amino acid 23 to about amino acid 350 of SEQ IDNO:2. The presence of the signal sequence, in addition to the fact thathDkk-3 has been identified using a Signal Sequence Trap system,indicates that hDkk:-3 is a secreted protein. Furthermore, theprediction of such a signal peptide and signal peptide cleavage site canbe made, for example, utilizing the computer algorithm SIGNALP (Nielsen,et al., (1997) Protein Engineering 10:1-6).

Examination of the cDNA sequence depicted in FIG. 1A-1, 1A-2, 1B-1, 1B-2shows that human Dkk-3 is particularly rich in cysteine residues. Asshown in FIG. 1A-1, 1A-2, 1B-1, 1B-2, hDkk-3 contains 20 cysteineresidues located between amino acid 147 and amino acid 284 of SEQ IDNO:2. This region has been termed the cysteine-rich region. Thesecysteine residues can form 10 disulfide bridges.

A BLAST search (Altschul et al., (1990) J. Mol. Biol. 215:403) of thenucleotide and the amino acid sequences of hDkk-3 has revealed thathDkk-3 is similar to a chicken cDNA encoding a protein of unknownfunction having GenBank Accession No. D26311. This cDNA was isolatedfrom a chicken lens cDNA library and was shown to be expressed in lensfibers and lens epithelium, but not in neural retina nor in liver cells.(Sawada et al., (1996) Int. J. Dev. Biol. 40:531), hDkk-3 and thechicken protein have 56% amino acid sequence identity and 72% amino acidsequence similarity. The amino acid sequence similarity between thechicken protein and human Dkk-3 is particularly high in thecysteine-rich domain of hDkk-3 which is located between amino acids 147and 284 of SEQ ID NO:2. In particular, the 20 cysteine residues ofhDkk-3 located in this region are present in the chicken protein.

Two genes recently identified in a screen for suppressors ofglioblastoma formation (Ligon et al. (1997) Oncogene 14:1075-1081) alsoshow homology to hDkk-3. These genes, RIG (“Regulated In Glioblastoma”)and RIG-like 7-1 (GenBank Accession Nos. U32331 and AF034208,respectively) were identified in a differential screen for mRNAsregulated by the introduction of a normal copy of chromosome 10 into aglioblastoma cell line harboring a deletion in chromosome 10 thatpromotes tumorigenesis. A schematic diagram summarizing the relationshipbetween the sequences of the hDkk-3 and the RIG genes is presented asFIG. 12. The indicated region of identity between hDkk-3 and RIGcomprises a short portion of the 3′ UTR of the human Dkk-3 mRNA (e.g.,RIG mRNA is ˜100% identical to residues 2479 to 2153 of SEQ ID NO:1).RIG-like 7-1 is homologous to hDkk-3 across a longer region (e.g., 97%identical from about nucleotides 316 to 2438 of SEQ ID NO:1) althoughthe encoded RIG-like 7-1 protein lacks the Dkk N-terminal signalsequence and is not therefore predicted to be a secreted protein. Thesedata associate hDkk-3 with human glioblastoma and suggest that hDkk-3may be important in the suppression of the tumorigenic phenotype. A rolein glioblastoma is also consistent with the high level of hDkk-3 mRNAexpression observed in human brain tissue. In addition. theco-localization of the hDkk-3, RIG and RIG-like genes to a region ofchromosome 11 (11p15.1) implicated in the development of human malignantastrocytoma (Ligon et al., supra) further indicates a role for thesegenes in tumorigenesis.

Human hDkk-3 protein has also some amino acid sequence similarity tometallothionein, particularly in the cyteine-rich domain.

Tissue Distribution of hDkk-3 mRNA

For Northern blots, all hybridizations were to Clontech Multiple TissueNorthern Blots and were performed in ExpressHyb solution (Clontech) for1-20 hours. All probes were prepared by random primed radiolabelling(Prime-It, Stratagene). Blots were washed sequentially to a finalstringency of 0.2×SSC/0.2% SDS and exposed to autoradiographic film.Hybridizations of a control β-actin cDNA probe consistently demonstratedeven loading of the Northern blots. The results of hybridization of theprobe to various mRNA samples are described below.

Hybridization of a Clontech Fetal Multiple Tissue Northern (MTN) blot(Clontech, LaJolla, Calif.) containing RNA from fetal brain, lung,liver, and kidney indicated the presence of high levels of hDkk-3 mRNA(˜2.5 kb) in fetal brain, lung, and slightly lower levels of hDkk-3 mRNAin fetal kidney. However, no significant level of hDkk-3 mRNA was foundin fetal liver.

Hybridization of a Clontech human Multiple Tissue Northern (MTN) blot(Clontech, LaJolla, Calif.) containing RNA from adult heart, brain,placenta, lung, liver, skeletal muscle, kidney, and pancreas with ahuman Dkk-3 probe indicated the presence of high levels of hDkk-3 mRNAin heart, slightly lower levels in brain, and much lower levels inplacenta and lung. Some hDkk-3 mRNA was also found in adult skeletalmuscle. However, no significant levels of hDkk-3 mRNA was observed inadult liver, kidney, or pancreas. Interestingly, the chicken gene whichis homologous to hDkk-3 was not expressed at detectable levels in livereither (Sawada et al., (1996) Int. J. Dev. Biol. 40:531).

Further hybridization of a Clontech human Multiple Tissue Northern (MTN)blot (Clontech, LaJolla, Calif.) including RNA from bone marrow, adrenalgland, trachea, lymph node, spinal cord, thyroid, and stomach revealedhigh levels of expression of hDkk-3 in mRNA isolated from adult spinalcord, and lower level expression in adrenal gland, trachea, thyroid, andstomach.

Thus, hDkk-3 is expressed in a tissue specific manner, with thestrongest expression observed in brain, heart, and spinal cord.

Example 2 Isolation and Characterization of mDkk-3 cDNA

In this example, the isolation and characterization of the gene encodingmurine Dkk-3 (also referred to as “mDkk-3”, “murine Cysteine RichSecreted Protein-1”, “murine CRSP-1” or “murine CRISPY-1”) is described.

Identification of a Murine Dkk-3 cDNA

A full length mDkk-3 cDNA was identified by comparison of the hDkk-3sequence to a proprietary EST Database using the BLAST-X algorithm. Asingle clone identified in a adult mouse brain cDNA library was obtainedand sequenced fully. DNA for the clone Fmmb059s was deposited with theATCC as Accession No. 98634. mDkk-3 is predicted to have a signalpeptide from residues 1 to 23 of SEQ ID NO:17, cleavage of which resultsin a mature protein having 326 amino acids in length corresponding toamino acids 24 to 349 of SEQ ID NO:17.

Tissue Distribution of mDkk-3 mRNA

To determine the expression pattern of mDkk-3, in situ hybridization wasperformed as follows. Normal mouse embryos and adult mouse tissues werecollected from C57BL/6 mice, embedded in TissueTek™ O.C.T Compound(Sakura Finetek U.S.A., Inc., Torrance, Calif.), frozen on dry ice, andstored at −80° C. Cryostat serial sections (8 μm) were thaw mounted onSuperfrost Plus™ slides (VWR Scientific, West Chester, Pa.) and airdried on a slide warmer at 40° C. for 20 minutes. Sections were thenfixed with 4% formaldehyde in DEPC treated 0.1 M phosphate-bufferedsaline (PBS. pH 7.5) at room temperature for 10 minutes and rinsed twicein DEPC-PBS. Sections were rinsed in 0.1 M triethanolamine-HCl (TEA, pH8.0), incubated in 0.25% acetic anhydride-TEA for 10 minutes and rinsedin DEPC-2×SSC (standard sodium citrate). Sections were dehydratedthrough a series of graded ethanols, incubated in 100% chloroform for 5minutes, rinsed in 100% and 95% ethanol for 1 minutes and air dried.

Antisense and sense RNA transcripts were prepared by in vitrotranscription (Riboprobe Gemini System™, Promega) of PCR amplified cDNAtemplates. Template amplification primers were as follows;

mDkk-3 forward 5′-CAGTGAGTGCTGTGGAGACC-3′, (SEQ ID NO: 30) and reverse5′-TCTTCAGTGCAGGCTCCTCTC-3′. (SEQ ID NO: 31)

Probes were labeled with ³⁵S-UTP (NEN) and purified on G-25 spin columns(Pharmacia). The hybridization cocktail contained: 50° % formamide, 10%dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodiumthiosulfate, 1×Denhardt's solution, 0.6 M NaCl, 10 mM Tris (pH 7.5), 1mM EDTA, 100 mM dithiothreitol (DTT), 0.1 mg/ml sheared salmon sperm, 50μg/ml yeast tRNA, 0.5 mg/ml yeast total RNA, and ³⁵S-UTP labeled probeat a concentration of 5×10⁷ c.p.m./100 μl of final hybridizationsolution; 100 μl of hybridization solution was put on each section. Thesections were then covered with a glass coverslip and incubated in ahumidified chamber at 55° C. for 18 h. After hybridization, slides werewashed with 2×SSC. Sections were then sequentially incubated at 37° C.in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and1 mM EDTA), for 10 minutes, in TNE with 10 ug/ml RNase A for 30 minutes,and finally in TNE for 10 minutes. Slides were then rinsed with 2×SSC atroom temperature, washed in 2×SSC at 50° C. for 1 h, 0.2×SSC at 55° C.for 1 h, and 0.2×SSC at 60° C. for 1 h. Sections were dehydrated with aseries of graded concentrations of ethanol 0.3 M ammonium acetate, airdried and exposed to Kodak Biomax MR™ scientific imaging film for 6 daysat room temperature.

mDkk-3 expression in the brain was found to be highly localized to thecortex and hippocampus but was not observed in the dentate gyrus. Higherpower magnification confirmed the mDkk-3 mRNA was localized to neuronswithin these structures. In the adult eye, mDkk-3 mRNA was found to behighly expressed in the retina, ciliary body, and lens epithelium.Expression in the retina was localized to the integrating bipolar andganglion cells. In adult heart, mDkk-3 was detected in theatrioventricular valves and also in myocytes of the atria. Expressionwas highly restricted to the atria and noticeably absent fromventricular tissue. High level expression of mDkk3 mRNA was alsoobserved in developing eye, bone and cartilage in day 14 embryos. Thesefindings corroborate and extend the northern analysis of hDkk-3 mRNAexpression in human tissues and also suggest that Dkk-3 may play a rolein bone and ocular physiology in addition to functions in neural andcardiac tissues.

Example 3 Secretion and Post-Translational Modification of Dkk-3

This example describes the secretion and post-translational modification(e.g., glycosylation and processing) of hDkk-3 as well as methods forsmall and large scale purification of hDkk-3.

hDkk-3 Expression Constructs

Expression constructs for two forms of hDkk-3 were prepared using themammalian expression vector pMET-stop. Form-1 comprised a cDNAincorporating the complete 350aa hDkk-3 protein coding sequence(hDkk-3flag.long) and form-2 comprised the entire hDkk-3 protein codingsequence except for the final 18 amino acids (hDkk-3 flag.short). AC-terminal sequence encoding the FLAG epitope (DYKDDDDK) (SEQ ID NO:19)was added to both hDlkk-3 forms for ease of detection and purification.hDkk-3flag cDNAs were generated by PCR from a full length hDkk-3 cDNAtemplate and ligated into pMET-stop using EcoRI and SalI restrictionsites.

Trial Transfection—Small Scale Expression

Expression constructs for hDkk-3flag.long and hDkk-3flag.short weretransfected into 293T cells using 10 μl of lipofectamine (GIBCO/BRL) and2 μg of DNA per well of a 6-well plate of cells which were 70-80%confluent. After 5 hours at 37° C., cells were fed with 1 ml of 20%FCS/DMEM. After incubation overnight at 37° C., cells were conditionedin 1 ml OptiMEM for 48 hours at 37° C. Samples of supernatant and cellpellets were solubilized in boiling SDS-PAGE gel buffer, run out on a4-20% SDS-PAGE gel, transferred to a nylon membrane and probed with theanti-FLAG monoclonal antibody M2. Samples from both supernatant andpellet samples showed significant immunoreactivity within a molecularweight range of 40-65 kDa on autoradiographic film using a HRPconjugated secondary antibody and ECL detection reagents. Thus, bothforms of hDkk-3 tested are secreted from 293T cells thereby confirmingexperimentally that hDkk-3 is a secreted protein. It should be notedthat the molecular weights of both forms of hDkk-3 tested are greaterthan predicted from the amino acid sequence, suggesting that the hDkk-3proteins secreted by 293T cells may be glycosylated. This is consistentwith the presence of four potential sites for N-linked glycosylation inthe hDkk-3 protein (e.g., at about amino acids 96-99, 106-109, 121-124,and 204-207 of SEQ ID NO:2).

Deglycosylation of hDkk-3

Given the heterogenous nature of secreted human Dkk-3, the effect ofN-Glycanase treatment on the mobility of secreted flag-tagged hDkk-3 wasstudied. Briefly, 1 mL samples of 293T cell supernatants collected 72hours after transfection with the appropriate constructs were incubatedwith 50 μL anti-flag M2 agarose beads (Sigma) for 16 hrs at 4° C. Beadswere washed with PBS (pH7.4) containing, sequentially, 0.1%, 0.05% and0.01% Triton X-100. The beads were resuspended in 20 μL of 20 mM sodiumphosphate, pH 7.5, 50 mM EDTA, 0.02% sodium azide, (incubation buffer)together with 0.5% SDS, 5% 2-mercaptoethanol and boiled for 2 minutes.The supernatant was split into equal 104, aliquots which were dilutedwith I0 μL incubation buffer, 5 μL 5% NP-40 and then with either 5 μLN-Glycanase (Oxford Glycosystems) in enzyme buffer (20 mM Tris-HCl, 1 mMEDTA, 50 mM NaCl, 0.02% sodium azide pH 7.5) or with enzyme buffer aloneas control. After 18 hours at 37° C., samples were boiled in equalvolumes of SDS-PAGE buffer and analyzed by SDS-PAGE and Westernblotting. For western analysis, samples were electroblotted onto PVDF(Novex) after SDS-PAGE on 4-20% gradient gels, probed with M2 anti-flagantibody (1:500, Sigma) followed by HRP conjugated sheep anti-mouse IgG(1:5000, Amersham), developed with chemiluminescent reagents(Renaissance, Dupont) and exposed to autoradiography film (Biomax MR2film, Kodak).

Utilizing the above-described methodology, it was determined that hDkk-3protein displayed a significant increase in mobility followingN-Glycanase treatment. The major 45-65 kD form of soluble hDkk-3 wasobserved as two species of 45-55 and 40 kD following deglycosylation.This finding is consistent with the presence of multiple potential sitesof N-linked glycosylation in the hDkk-3 protein. The reason for theheterogeneity of deglycosylated hDkk-3 reflects either proteolyticprocessing or incomplete removal of carbohydrate from one or moreattachment sites. A30 kD hDkk3 species was also observed in theseexperiments, the mobility of which was unaltered by N-Glycanasetreatment. This form was only observed after overnight incubation of thesamples and may be a non-specific degradation product.

Large Scale hDkk-3 Protein Production

For scale-up of hDkk-3flag.long protein expression, 30×150 mM plates of293T cells at 70-80% confluence were transfected with 27 μg DNA. 100 μllipofectamine in 18 ml OptiMEM for 5 hours at 37° C. 18 ml of 10%FCS/DMEM was added to each plate and incubated overnight at 37° C., 24hours after the start of transfection, transfection supernatant wasaspirated and 35 mls OptiMEM was added to each plate and the platesincubated at 37° C. for 72 hours. Conditioned medium was harvested spunat 4000 rpm for 30 min. at 4° C., and filtered through a 0.45 micronfilter unit. 1100 ml was passed over a 1.6×10 cm anti-FLAG M2 affinitycolumn pre-equilibrated in PBS pH7.4 buffer at a flow rate of 2.0 ml perminute. After washing with 200 ml of PBS pH 7.4 buffer, bound materialwas eluted by a step of 200 mM Glycine pH 3.0 buffer and 0.5 mlfractions collected. Upon elution, a significant protein peak wasdetected by absorbance at 280 nm. Samples corresponding to conditionedmedium, flow through and eluted fractions were analyzed by Coomassieblue and silver stained SDS-PAGE and by western blot analysis asdescribed above. Significant immunoreactivity within a molecular weightrange of 40-65 kDa was detected in conditioned medium and elutedfractions but not in the flow through sample, indicating that thesecreted hDkk-3flag.long protein bound to the affinity columnspecifically and was eluted efficiently by the described conditions.Coomassie blue staining of SDS-PAGE gels suggested that the predominantimmunoreactive protein constituted >90% of the protein present in thebound and eluted protein peak. Peak fractions of eluted protein werepooled and dialysed against Phosphate Buffered Saline resulting in a 4ml volume of recombinant hDkk-3flag.long protein at a concentration ofapproximately 1 mg/ml.

Example 4 Isolation and Characterization of hDkk-4

In this example, the isolation and characterization of the gene encodinghuman Dkk-4 (also referred to as “hDkk-4”, “Cysteine Rich SecretedProtein-2”, “CRSP-2” or “CR1SPY-2”) is described.

Isolation and Analysis of a Human Dkk-4 cDNA

To identify novel proteins related to hDkk-3, the human Dkk-3 amino acidsequence was used to search the dbEST database using TBLASTN(WashUversion. 2.0, BLOSUM62 search matrix). A dbEST clone withaccession number AA565546 was identified as having homology to a portionof the hDkk-3 cDNA. This clone was obtained from the IMAGE consortiumand sequenced fully to define the entire hDkk-4 sequence depicted inFIG. 2A-2B.

Determination of the hydrophobicity profile of human Dkk-4 having theamino acid sequence set forth in SEQ ID NO:5 indicated the presence of ahydrophobic region from about amino acid 1 to about amino acid 19 of SEQID NO:5. Further analysis of the amino acid sequence SEQ ID NO:5 using asignal peptide prediction program predicted the presence of a signalpeptide from about amino acid 1 to about amino acid 19 of SEQ ID NO:5.Accordingly, the mature hDkk-4 protein includes about 205 amino acidsspanning from about amino acid 20 to about amino acid 224 of SEQ IDNO:5.

Tissue Distribution of hDkk-4

hDkk-4 mRNA was undetectable by Northern analysis in all adult and fetalhuman tissues examined. Accordingly, a survey was performed of a cDNAlibrary panel by PCR with hDkk-4 specific PCR primers. Using suchprimers, products were identified in libraries prepared from cerebellum,activated human T-lymphocytes, lung and esophagus.

Secretion and Post-Translational Modification of human Dkk-4

Flag epitope-tagged human Dkk-4 protein was transiently overexpressed in293T cells and analyzed as described previously for hDkk-3. SolublehDkk-4 was consistently detected as three major immunoreactive speciesof approximately 40 kD [form (i)], 30-32 kD [form (ii)] and 15-17 kD[form (iii)]. Neither form (i), (ii) or (iii) was significantly affectedby N-glycanase treatment, consistent with the absence of N-glycosylationsites from the protein.

To determine the possible cause of heterogeneity in the size of secretedhDkk-4, Edman N-terminal sequencing of anti-flag affinity purifiedmaterial corresponding to bands (i), (ii) and (iii) was performed.Briefly, flag-tagged Dkk-4 protein was isolated by passing theconditioned media over an M2-biotin (Sigma)/streptavidin Poros column(2.1×30 mm, PE Biosystems); the column was then washed with PBS, pH 7.4and flag-tagged protein eluted with 200 mM glycine, pH 3.0. Elutedfractions with 280 nm absorbance greater than background were analyzedby SDS-PAGE and western blot. Purified Dkk-4 protein bound to PVDFmembrane after SDS-PAGE and electroblotting was sequenced for N-terminalamino acid analysis on a PE Applied Biosystems Model 494 Preciseinstrument using Edman-based chemistry protein sequencing. The aminoacid residues were analyzed by HPLC (Spherogel micro PTH 3-microncolumn) and determined by separation and peak height as compared tostandards.

The N-terminal sequence of band (i) was found to be XVLDFNNIRS (SEQ IDNO:34) which corresponds exactly to the predicted signal peptidecleavage site (between Ala-18 and Leu-19). Because the same band isidentified by anti-flag antibodies, which recognize the C-terminalepitope tag, band (i) was thus identified as the full length, maturehDkk-4 protein. The band (iii) N-terminal sequence was found to beSQGRKGQEGS (SEQ ID NO:38) which corresponds to CRD-2 cleaved at thedibasic site Lys132/Lys133 (e.g., Lys/113/Lys114 of the mature proteinfollowing cleavage of the a 19 amino acid signal sequence or Lys 114/Lys115 following cleavage of a 18 amino acid signal sequence). These dataobtained for bands (i) and (iii) indicate clearly that hDkk4 isproteolytically processed by 293T cells, resulting in the release ofCRD-2 (a 91 amino acid biologically-active fragment) from the fulllength protein.

Moreover, the three major species migrated similarly on SDS-PAGEconducted under either reducing or non-reducing conditions. Thus, eachof the major C-terminal (anti-flag immunoreactive) hDkk-4 species existas independent proteolytic fragments that are not covalently linked viadisulfide bonds to other subunits or complex components when secretedfrom 293T cells.

Example 5 Isolation and Characterization of hDkk-1

In this example. the isolation and characterization of the gene encodinghuman Dkk-1 (also referred to as “hDkk-1”, “Cysteine Rich SecretedProtein-3”, “CRSP-3” or “CRISPY-3”) is described.

Identification of a Human Dkk-1 cDNA

Searching a proprietary database of EST information using the sequenceof hDkk-3, an hDkk-1 partial sequence was found corresponding to a clonefrom a human fetal kidney cDNA library having the identification codejthKb075a10. This clone was sequenced further and to define the entirehDkk-1 sequence depicted in FIG. 3A-3B. DNA for the clone jthKb075a10was deposited with the ATCC as Accession No. 98633. hDkk-1 has apredicted signal peptide from about amino acid residue 1 to 20 of SEQ IDNO:8, cleavage of which results in a mature protein having 246 aminoacid residues in length and corresponding to amino acid residues 21 to266 of SEQ ID NO:8.

Tissue Distribution of hDkk-1

Northern blot analysis of various tissues including heart, brain,placenta, lung, liver, skeletal muscle, kidney, and pancreas wasperformed as previously described using a probe specific for hDkk-1. A˜1.8 kb hDkk1 mRNA was detected at high levels in human placenta, butnot in other tissues tested.

Secretion and Post-Translational Modification of hDkk-1

Flag epitope-tagged human Dkk-1 protein was transiently overexpressed in293T cells and analyzed as described previously. hDkk-1 was efficientlysecreted from mammalian cells and was readily detected in conditionedmedium of transfected cells. Mature secreted hDkk-1 migrated with amolecular weight of approximately 42-50 kD. Treatment with N-Glycanasehad no significant effect on the mobility of soluble hDkk-1. AlthoughhDkk-1 contains one potential site of N-linked glycosylation at itsextreme C-terminus (e.g., at amino acids 256-259 of SEQ ID NO 8), thissite is not conserved in Xenopus Dkk-1 (Glinka et al., supra) andappears not to be a major site of carbohydrate addition in 293T cells.

Example 6 Isolation and Characterization of hDkk-2

In this example, the isolation and characterization of the gene encodinghuman Dkk-2 (also referred to as “hDkk-2”, “Cysteine Rich SecretedProtein-4”, “CRSP-4” or “CRISPY-4”) is described.

Isolation of a Human Dkk-2 cDNA

Using the hDkk-3 sequence to query the dbEST database, a clone havingsimilarity to a portion of hDkk-3 was identified having Accession No.W55979. This clone was subsequently obtained from the IMAGE consortiumand sequenced to define a partial hDkk-2 sequence set forth as SEQ IDNO:10. This cDNA comprises a coding region from nucleotides 1-537, aswell as 3′ untranslated sequences (nucleotides 538 to 702). The codingregion alone is set forth as SEQ ID NO:12. The predicted amino acidsequence corresponds to amino acids 1 to 179 of SEQ ID NO:11. A cDNAencoding full length hDkk-2 was isolated from a human fetal lung lambdaZiplox libraries by conventional plaque hybridization (Sambrook et al.,1989) and fully sequenced. The full-length nucleotide sequence is setforth as SEQ ID NO:20 and the predicted amino acid sequence is set forthas SEQ ID NO:21. The coding region alone is set forth as SEQ ID NO:22.The predicted amino acid sequence corresponds to amino acids 1 to 259 ofSEQ ID NO:21. DNA for the clone fthu133 was deposited with the ATCC asAccession No. 207140. hDkk-2 has a predicted signal peptide from aboutamino acid residue 1 to 33 of SEQ ID NO:21, cleavage of which results ina mature protein having 226 amino acid residues in length andcorresponding to amino acid residues 34 to 259 of SEQ ID NO:21.

Tissue Distribution of hDkk-2

Northern blot analysis of various tissues (e.g., heart, brain, skeletalmuscle, colon. thymus. spleen, kidney, liver, small intestine, placenta,lung. and peripheral blood leukocytes) was performed as previouslydescribed using a probe specific for hDkk-2. Of the tissues tested.hDkk-2 mRNA expression was highest in heart. brain, placenta, lung, andskeletal muscle. hDkk-2 transcripts of approximately 4.0 and 4.5 kb wereobserved.

Secretion and Post-translational Modification of hDkk-2

Flag epitope-tagged human Dkk-2 protein was transiently overexpressed in93T cells and analyzed as described previously. Soluble hDkk-2 wasdetected as a major species of 15-17 kD, closely similar in size to form(iii) of hDkk-4. Additional minor forms of hDkk-2 were also observed incertain experiments in the range of 20-21 kD. Deglycosylation of hDkk-2was not studied since the protein sequence lacks potentialN-glycosylation sites. By comparison with the data presented in Example4 regarding the dibasic proteolytic cleavage site in the hDkk-4 proteinsequences, it is predicted that the major 15-17 kD form of hDkk-2detected in these experiments corresponds to CRD-2, as was the case forhDkk-4.

Example 7 Isolation of Soggy Proteins

In this example, the isolation and characterization of the gene encodinghuman and murine Soggy-1 (also referred to as “Cysteine Rich SecretedProtein-N” or “CRISP-N”) is described.

Identification of a Human and Murine Soggy-1 cDNAs

Human Soggy-I was identified as a novel protein with similarity to theN-terminal domain of hDkk3. A human partial sequence was identified inthe dbEST database for a clone having the accession number AA397836.This clone was obtained from the IMAGE collection and sequenced fully todefine the entire human Soggy-1 sequence depicted in FIG. 7A-7B. Twomurine partial sequences were likewise identified in the dBEST database.The clones were obtained from the IMAGE consortium and sequenced. Theentire murine Soggy-I sequence is depicted in FIG. 8A-8B. Human andmurine Soggy cDNAs encode proteins of 242aa and 230aa, respectively, andare predicted to be secreted owing to the presence of N-terminal signalpeptides. hSoggy-1 has a predicted signal peptide from about amino acidresidue 1 to 30 of SEQ ID NO:14, cleavage of which results in a matureprotein having 194 amino acid residues in length and corresponding toamino acid residues 31 to 224 of SEQ ID NO:14. mSoggy-1 has a predictedsignal peptide from about amino acid residue 1 to 20 of SEQ ID NO:27,cleavage of which results in a mature protein having 210 amino acidresidues in length and corresponding to amino acid residues 21 to 230 ofSEQ ID NO:27. Human and murine Soggy proteins display 59% overallidentity although significant amino acid identities are seen beyond thisdomain that extend into the CRDs of Dkk-3 (FIG. 10A-10B). However,cysteine residues are not conserved within these domains and theresidues shared by Soggy and Dkk-3 are poorly conserved in other Dkksindicating that the sequence relationship between these proteins isunique. Homology is most obvious within a 51 amino acid region in which33% identity is observed between hSoggy, mSoggy, hDkk-3 and mDkk-3. This51 amino acid domain is referred to herein as an SGY domain. Human andmouse Soggy-1 proteins each possess 2 sites of potential N-linkedglycosylation which are within the SGY domain and are also conservedwith Dkk3. (e.g., NNTL, corresponding to amino acid residues 97-100 ofSEQ ID NO:14 and NKTG corresponding to amino acid residues 112-115 ofSEQ ID NO:14). In contrast to other Dkks, the C-terminal domain ofSoggy-1 shows no similarity to other protein sequences in the publicdatabases nor does it contain any cysteine residues. Soggy was so namedin view of its lack of CRDs compared to hDkk-3, which had beenpreviously designated Cysteine Rich Secreted Protein-1 (“CRISPY-1”).

Tissue Distribution of Soggy-1

To investigate Soggy-1 mRNA expression, a mouse cDNA probe was used onmurine Northern blots. A 1 kb mSoggy-1 mRNA was expressed at very highlevels in testis and, interestingly, demonstrated transient expressionduring mouse embryogenesis. Soggy-1 mRNA, which was undetectable at day7 of gestation, was transiently expressed at day 11 and day 15, afterwhich the expression level declined to undetectable levels. Thus,mSoggy-1 displays a developmentally regulated pattern of expression.

In situ analysis was performed as described in Example 1. For detectionof 25 murine Soggy-1, the following primers were used:

mSoggy forward 5′ ACCTGCAATGTGTCGACTGAG-3′, (SEQ ID NO: 32) and reverse5′-CACTTACAGCTGTTGGGATG-3′. (SEQ ID NO: 33)

Consistent with the Northern analysis, very high level expression ofSoggy-1 mRNA was observed by in situ analysis in adult testis. Upon highmagnification, Soggy-1 mRNA was found to be expressed at high levels inthe spermatogenic epithelium of the seminiferous tubules and in thespermatogonia at various stages of development. A series of saggitalsections of mouse embryos from E13.5-E 17.5 and post-natal day 1.5 pupswere also analyzed. In E15.5 embryos, Soggy-1 mRNA transcripts werelocalized to the developing dorsal root ganglia (DRGs) and also found inthe cartilage primordium of the nasal septum. Soggy-1 expression wasalso seen in the eve from E 13.5 to E 16.5, as observed for mDkk-3.Expression of Soggy-1 mR.NA at various stages of development isconsistent with the northern analysis described above and suggests thatSoggy-1 may play a role in multiple stages of development.

Secretion and Post-Translational Modification of Soggy Proteins

Flag epitope-tagged human Soggy-1 protein was transiently overexpressedin 293T cells and analysed as previously described. hSoggy wasefficiently secreted from transfected 293T cells and migrated with amolecular weight of approximately 40-50 kD. Given the heterogenousnature of secreted human Soggy-1, the effect of N-Glycanase treatment onthe mobility of secreted flag-tagged hSoggy-1 was studied. hSoggydisplayed a 5-10 kD decrease in apparent molecular weight afterN-Glycanase treatment, consistent with the presence of 2 potential sitesof N-glycosylation in the protein.

Example 8 Structure of the Dkk Family Proteins and Dkk-Related Proteins

The amino acid and nucleotide homology between Dkk family members andDkk-related proteins is set forth in the following tables. Whereindicated, mDkk-1 and xDkk-1 correspond to a murine and Xenopus proteinsset forth in Glinks et al., supra, and having Accession Nos: AF030433and AF030434, respectively. Likewise cDkk-3 has Accession No. D26311

Table II sets forth overall sequence identities as determined using theALIGN program, (version 2.0) using a PAM 120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4:

hDkk-3 hDkk-4 hDkk-1 hDkk-2 mDkk-1 xDkk-1 CLFEST hDkk-3 100 16.0 18.615.1 18.5 16.5 53.0 hDkk-4 100 33.7 35.2 32.6 33.7 16.2 hDkk-1 100 33.180.2 53.5 17.4 hDkk-2 100 30.5 33.7 12.5

Table III sets forth nucleic acid identities as determined using theusing the Wilbur Lipman DNA alignment program, Ktuple: 3; Gap Penalty:3; Window: 20:

hDkk-3 hDkk-4 hDkk-1 hDkk-2 mDkk-1 xDkk-1 CLFEST hDkk-3 100 30.0 37.234.7 31.5 45.4 58.8 hDkk-4 100 43.0 35.9 38.8 38.4 36.7 hDkk-1 100 59.366.4 53.7 32.1 hDkk-2 100 38.8 38.4 36.7

Table IV sets forth local amino acid identities as determined using theSmith-Waterman algorithm as implemented in the program Bestfit of theGCG package, with gap penalties of 8 for opening and 1 for extending:

hDkk-1 mDkk-1 82 xDkk-1 64 63 hDkk-2 50 48 47 hDkk-3 39 37 37 37 mDkk-336 33 38 40 83 cDkk-3 34 31 35 36 61 60 hDkk-4 45 43 47 46 40 36 34hDkk-1 mDkk-1 xDkk-1 hDkk-2 hDkk-3 mDkk-3 cDkk-3 hDkk-4

A multiple alignment of the amino acid sequences of hDkk-1, hDkk-2,hDkk-3, hDkk-4, mDkk-1, mDkk-3, xDkk-1, and cDkk-3 is shown in FIG.6A-6C. Predicted signal peptides are underlined, N-glycosylation sitesare indicated by a thick bar. CRD-1 by an open box and CRD-2 by a shadedbox. The proteolytic cleavage site within hDkk4 is indicated by a doubleasterisk. The domain structure of the full length human Dkk proteins ofthe present invention as well as human Soggy are schematicallyillustrated in FIG. 9. Signal peptides (darkened boxes), Cysteine RichDomain 1 (“CRD-1”) (also referred to as the “amino-terminalcysteine-rich domain”). Cysteine Rich Domain 2 (“CRD-2”) (also referredto as the “carboxy-terminal cysteine-rich domain”), the soggy domain(SGY) within hDkk-3 and hSoggy-1, and sites of N-glycosylation areindicated.

As demonstrated at least in FIGS. 6A-6C and 9, human Dkks 1 through 4each possess an N-terminal signal peptide and contain two conservedcysteine-rich domains (CRDs) separated by a linker or spacer region.Each CRD possesses 10 conserved cysteine residues. The second CRD haselsewhere been described as a colipase-like domain because the positionsof the ten conserved cysteines in this domain have been shown to beclosely similar to those in proteins of the colipase family (Aravind andKoonin, supra). Conservation of CRD-1 and CRD-2 suggests importantfunctions for these domains. In contrast to the CRDs, the linker orspacer region that joins CRD-1 and CRD-2 is highly variable betweenhDkks, being notably larger in hDkk-1, -2 and -4 (50-55aa) as comparedto Dkk-3 (12aa). Four potential sites of N-linked glycosylation arepresent in hDkk3 and are conserved in chicken and mouse Dkk-3. Thesesites are not conserved in other Dkk family members. hDkk1 possesses onepotential N-glycosylation site located close to the C-terminus of theprotein which is conserved in murine Dkk-1 but not in Xenopus Dkk-1(FIG. 6A-6C). In addition, each hDkk possesses several potential dibasicproteolytic cleavage sites, suggesting the proteins may be subject topost-translational processing. hDkk3 is the most divergent of the fourhuman Dkks and possesses an extended N-terminal unique region whichprecedes CRD-1 and an extended C-terminal unique region which is highlyacidic.

Example 9 Effects of hDkks and Soggy on Wnt-Induced Axis Duplication inXenopus Embryos

This Example describes the functional activities of the hDkk and Soggyproteins of the present invention.

Xenopus Embryo Culture and RNA Microinjections

Eggs were obtained from Xenopus females injected with 700 units of humanchorionic gonadotropin, fertilized in vitro and cultured in 0.1×MMR(Newport and Kirschner (1982) Cell 30:675-686). Embryonic stages weredetermined according to Nieuwkoop and Faber (1967) Normal table ofXenopus laevis (Daudin) Amsterdam: North Holland Publ. All cDNAs weresubcloned into pCS2 vector (Rupp et al. (1984) Genes & Development8:1311-1323), and capped mRNAs were synthesized in vitro as described(Krieg and Melton (1984) Nucleic Acids Res. 12:7057-7070, using theMessage Machine kit (Ambion). The following plasmids were used astemplates for mRNA synthesis: hDkk-1-pCS2, hDkk-2-pCS2, hDkk-3-pCS2,hDkk-4-pCS2, hSoggy-pCS2, Xwmt8 (Christian et al., (1991) Development111:1045-1055), Xwnt2B (Landesman and Sokol (1997) Mech. Dev.61:1199-209), Xwnt3a (Wolda et al. (1993) Dev. Biol. 155:46-5),Xfz8-pXT7 (Itoh et al. (1998) Mech. Devel. 74:145-157), Xdsh-pXT7(Sokol, et al. (1995) Mech. Devel. 74:145-157). Protein expression fromall pCS2-Dkk constructs was confirmed by in vitro transcription andtranslation (TNT, Promega). For secondary axis induction, a singleventral blastomere of 4- or 8-cell embryos was injected with 10 nl of asolution containing 2-4 pg of Xwnt8 mRNA, 2.5-5 pg of Xwnt3a mRNA or 10pg of Xwnt2B mRNA as described (Itoh et al, (1995) supra.). The effectof Dkk RNAs was tested by coinjecting Wnt mRNAs with 2.5 ng of hDkkmRNAs. For studies of Frizzled and Dhshevelled, 5 ng Fz8 and 1 ng XdshmRNAs were injected as indicated. After injections, embryos werecultured in 3% Ficoll 400 (Pharmacia), 0.5×MMR. Secondary axes werescored at stage 35 as complete, when they contained anteriorneuroectodermal derivatives including pronounced cement gland and eyes,and as partial, when the secondary neural tube with melanocytes, butwithout head structures, was apparent.

Inhibition of Secondary Axis Induction by hDkk-1 and hDkk-4 in XenopusEmbryos

hDkk-1 or hDkk-2 mRNAs were coinjected with Xwnt8 mRNA into singleventral blastomeres of 4- or 8-cell embryos. Injected embryos werecultured for 2 days and secondary axes were scored based on externalmorphology. Xwnt8 injected embryos displayed complete axis duplication,which was inhibited by co-injection with mRNAs encoding hDkk-1 andhDkk-4. To determine whether hDkks interacted with specific Wnt ligands,several different Wnts were assayed in combination with hDkk-1 or hDkk-4for secondary axis formation. hDkk-1 and hDkk-4 inhibited axisduplication in response to Xwnt3a and Xwnt2b in addition to Xwnt8.hDkk-1 consistently demonstrated stronger inhibition of Wnt signalingthan hDkk-4. Thus, hDkk-1 and hDkk-4 do not show any clear selectivityfor the Wnt ligands used in this study. This compares to the FRPs, whichalso show little specificity with respect to their ability to inhibitWnts (Leyns et al. (1997) supra; Wang et al. (1997) supra; Salic et al.(1997) supra; Mayr et al. (1997) supra; Finch et al. (1997) supra).

To investigate the mechanism by which hDkk-1 and hDkk-4 inhibit Wntsignaling, Dkk mRNAs were coinjected with Xdsh, a downstream componentof the Wnt signaling pathway (Itoh et al. (1998) supra). hDkks-1 and -4did not block secondary axis formation by Xdsh, indicating that Dkksfunction upstream of, or parallel with, Xdsh signaling. Similar findingshave been reported previously for xDkk-1 (Glinka et al. (1998) supra).It was also determined whether hDkks could antagonize signaling byXenopus Frizzled-8 (Xfz8), which can also induce a secondary axisthrough Wnt signaling (Itoh et al. (1998) supra). Neither hDkk-1 orhDkk-4 inhibited the axis-inducing activity of Xfz8 mRNA. This data,taken together with the fact that hDkk-1 and hDkk-4 are secreted,indicate that Dkks antagonize Wnt signaling at a point upstream of Wntreceptors.

Assay for Inhibition of Secondary Axis Induction by hDkk-2, hDkk-3 andhSoggy-1 in Xenopus Embryos

hDkk-2, hDkk-3 or Soggy mRNAs were coinjected with Xwnt8 mRNA intosingle ventral blastomeres of 4- or 8-cell embryos and secondary axeswere scored after two days as described for hDkk-1 and hDkk-4. Injectionof mRNAs encoding hDkk-2, hDkk-3 or hSoggy-1 had no effect onXwnt8-induced axis duplication. The ability of hDkk-2, hDkk-3 andhSoggy-1 to interact with specific Wnt ligands was also determined asdescribed previously. hDkk-2, hDkk-3 and hSoggy-1 were inactive againsteach of the three Wnts tested. The lack of activity of hDkk-2, hDkk-3and hSoggy-1 suggests that these proteins antagonize other members ofthe Wnt superfamily not tested here, or that they perform functionsdistinct from Wnt inhibition.

Example 10 Preparation of Antibodies Specific for hDkk and hSoggyProteins

This example describes the making of polyclonal antibodies specific forhDkk-1, hDkk-4, hDkk-1 hDkk-2, and hSoggy-1.

Peptides were synthesized using Fmoc solid phase methodology utilizingMAP resin technology which increases the antigenic response (Tarn (1988)Proc. Natl. Acad. Sci. USA 85:5409-5413. For each protein, the peptidesused for immunization 20 are listed below:

hDkk-3 peptide #44 (amino acids 51-65 of SEQ ID NO: 2) FREVEELMEDTQHKLpeptide #46 (amino acids 311-325 of SEQ ID NO: 2) GSFMEEVRQELEDLE hDkk-4peptide #91 (amino acids 111-125 of SEQ ID NO: 5) HAEGTTGHPVQENQP hDkk-1peptide #93 (amino acids 71-85 of SEQ ID NO: 8) GNKYQTIDNYQPYPC hDkk-2peptide #56 (amino acids 73-87 of SEQ ID NO: 11) GHYSNHDLGWQNLGRhSoggy-1 peptide #58 (amino acids 197-211 of SEQ ID NO: 14)LQAIRDGLRKGTHKD

Peptides were designed to meet at least the following criteria: (1) notincluded within the cysteine-rich domain; (2) not including anN-glycosylation site; and (3) hydrophilic (e.g., solvent exposed).

Antibodies were generated in New Zealand white rabbits over a 10-weekperiod. The immunogen includes KLH-peptide emulsified by mixing with anequal volume of Freund's Adjuvant, and injected into three subcutaneousdorsal sites, for a total of 0.1 mg peptide per immunization. Animalswere bled from the articular artery. The blood was allowed to clot andthe serum collected by centrifugation. The serum is stored at −20° C.

For purification, peptide antigens were immobilized on an activatedsupport. Antisera was passed through the sera column and then washed.Specific antibodies were eluted via a pH gradient, collected and storedin a borate buffer (0.125M total borate) at ˜0.25 mg/ml. Theanti-peptide titers were determined using ELISA methodology with freepeptide bound in solid phase (1 μg/well). Detection was obtained usingbiotinylated anti-rabbit IgG, HRP-SA conjugate, and ABTS.

All antibodies performed well in ELISA assays. Anti-peptide #44, #46,and #58 are particularly useful for detection of hDkk-3 and hSoggy-1,respectively, as determined by western blotting of supernatants fromhDkk-3- and hSoggy-1-transfected 293T cells.

The Dkk family comprises a novel family of secreted proteins which todate includes hDkk-1, hDkk-2, hDkk-3, hDkk-4, xDkk-1, mDkk-1 and cDkk-1.Structurally, Dkks 1-4 are related by several conserved features.Firstly, all four proteins are secreted proteins. Secondly, Dkks 1-4each possess two distinct cysteine rich domains. Each domain contains 10conserved cysteine residues, and these domains are highly conservedbetween family members. The C-terminal cysteine rich domain, referred toas CRD-2, bears significant similarity to proteins of the colipasefamily and sequence conservation among the Dkks is greatest within CRD-2(Aravind and Koonin, supra). This may reflect a need for Dkks tointeract with lipids in order to regulate Wnt function, since Wntproteins remain tightly associated with the cell surface.

Despite the similarities between Dkks 1-4, notable differences betweenthese family members appear with regard to their mRNA expressionpatterns. In adult human tissues hDkk-1 and hDkk-4 showed highlyrestricted mRNA expression patterns while hDkk-2 and hDkk-3 are morewidely expressed. Murine Dkk-3 mRNA was found to be restricted to themyocytes of the atria in the heart, neurons of the cortex andhippocampus in the brain and also to the retinal neurons and lensepithelium in the eye. Such specific expression patterns reflectlocalized action of the Dkks as regulators of Wnt activity and/or thatof other signaling molecules. Different Wnt family members have beenshown to have divergent patterns of mRNA expression in adult andembryonic mammalian tissues. For example, murine Wnts 4, 7a and 7b areexpressed in brain and lung, whereas Wnt6 is highly expressed in testis(Gavin et al., (1990). Wnts 5b and 13 are more broadly expressed (Gavinet al. (1990) supra; Katoh et al. (1996) supra). Although Wnts have beenstudied mostly in the context of their roles in embryonic developmentand tumorigensis, the expression of many family members in normal adulthuman and mouse tissues, together with their regulators such as theDkks, suggests that these signaling proteins play important roles innormal tissue homeostasis.

Marked differences in the post-translational processing of differenthuman Dkk proteins was also observed. hDkk-3 is secreted from 293T cellsas a heterogeneously glycosylated protein, whereas Dkk-1, 2 and 4proteins show no evidence of glycosylation. This is consistent withsequence analysis that identifies 4 potential sites of N-glycosylationin the hDkk-3 protein but no sites in either hDkk-2 or hDkk-4. A singleputative site in hDkk-1 does not appear to be utilized in 293T cells andmay well not be a significant site for N-linked carbohydrate addition inview of its C-terminal location and lack of conservation with xDkk-1.C-terminal proteolysis of hDkk4 in 293T cells was also characterized.Dkk proteins contain multiple potential sites of proteolytic processing.Secreted hDkk-4 was consistently detected as three major C-terminalfragments. N-terminal sequencing identified two of these as mature, fulllength hDkk4 and CRD-2, which was derived from the full length proteinby a specific proteolytic event at lysines 132 and 133. Thus, the hDkk-4CRD-2 is released from the full length protein upon expression in 293Tcells. Similar processing of hDkk4 in COS cells has been observed.

Within the Dkk family, Dkks 1, 2 and 4 display closest similaritywhereas Dkk-3 is more distantly related. For example, Dkk-3 contains alinker region connecting CRD-1 and CRD-2 which is shorter than in otherDkks. Dkk-3 also possesses extended N- and C-terminal regions comparedto other Dkks. Within the Dkk-3 N-terminal unique region, a distinctdomain has been identified that is also found in Soggy (the SGY domain).The SGY domains of human and mouse Soggy-1 and Dkk-3 proteins containtwo conserved sites of N-linked glycosylation. Protein expressionstudies confirm that, like hDkk3, hSoggy is secreted as a glycoprotein.Murine Soggy-1 is highly expressed in adult testis and also displays ahighly restricted mRNA expression in E15-E16 mouse embryos, beinglocalized mainly to the cartilage primordia within the developingvertebrae/developing dorsal root ganglia. Soggy mRNA was also detectedat high levels in the developing eye, similar to Dkk-3. Thisdevelopmentally regulated pattern of Soggy expression overlaps with thatof Dkk-3 suggesting that Soggy may play a role in the regulation ofDkk-3 function.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide comprising at least 100 contiguous aminoacids of SEQ ID NO:11.
 2. The isolated polypeptide of claim 1, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:11. 3.The polypeptide of claim 1 further comprising at least one heterologousamino acid sequence.
 4. An isolated polypeptide encoded by thepolynucleotide of SEQ ID NO:10, wherein said polypeptide comprises atleast 100 contiguous amino acids of SEQ ID NO:11.
 5. The isolatedpolypeptide of claim 1 comprising the polypeptide of SEQ ID NO:21.
 6. Amethod for identifying a compound which binds to a polypeptide of claim1 comprising: (a) contacting a polypeptide of claim 1, or a cellexpressing said polypeptide with a test compound; and (b) determiningwhether the polypeptide binds to the test compound.
 7. The method ofclaim 6 wherein the binding of the test compound to the polypeptide isdetected by a method selected from the group consisting of: (a)detection of binding by direct detection of test compound/polypeptidebinding; and (b) detection of binding using a competition binding assay.